Polymer Precision Filtration Device

Abstract

To provide unique filter holder designs and constitutions and methods appropriate to hold microfilters to collect rare cells and allow performing assays in the filter holder.SOLUTION: A microfilter 1520 comprises a polymer layer formed from epoxy-based photo-definable dry film, and a plurality of apertures each extending through the polymer layer. Rare cells are collected from body fluids to perform assays. The rare cells collected on the microfilter can be used for medical and biological research applications.SELECTED DRAWING: Figure 15

Description

Technical Field

[0001] The present invention generally relates to a precision filtration device including a polymer microfilter, a method for manufacturing the same, a method for using the precision filtration device, and an application of the device.

Background Art

[0002] Some medical conditions can be diagnosed by detecting the presence of certain types of cells in body fluids. In particular, the cells characteristic of or indicative of a particular medical condition may be larger and / or less flexible than other cells found in certain body fluids. Thus, it is also possible to diagnose a medical condition based on collected cells by collecting the larger and / or less flexible cells from a liquid sample such as a body fluid.

[0003] Cells that are larger and / or less flexible than other cells present in a body fluid can be collected by filtering the body fluid. For example, target cells indicative of a condition can be collected by passing the body fluid through a filter having an aperture sized too small for the target cells to pass through but large enough for other cells to pass through. Once collected, any number of analyses of the target cells can be performed. Such analyses can include, for example, identifying, counting, characterizing, and / or culturing the collected cells.

[0004] Microfilters desirably have accurate pore dimensions, do not break during use, and are not autofluorescent for fluorescence microscopy imaging. Conventionally, microfilters are installed in a precision filtration device and the liquid sample being processed to collect cells based on size on the microfilter.

[0005] <Cross - reference to Related Applications> This application is a partial continuation application of patent application PCT / US No. 11 / 30996, "Polymer Microfilter and Method of Manufacturing the Same", filed on April 1, 2011.

[0006] This application also claims priority to U.S. Provisional Patent Application No. 61 / 562404, filed on 21 November 2011, “Polymer Microfiltration Apparatus, Method for Manufacturing the Same and Use of the Microfiltration Apparatus,” the entirety of which is incorporated herein by reference.

[0007] This application also claims priority to U.S. Provisional Patent Application No. 61 / 618,641, filed on March 30, 2012, “Polymer Microfiltration Apparatus, Method for Manufacturing the Same and Use of the Microfiltration Apparatus,” the entirety of which is incorporated herein by reference.

[0008] This application also claims priority to U.S. Provisional Patent Application No. 61 / 654,636, filed on June 1, 2012, “Polymer Microfiltration Apparatus, Method for Manufacturing the Same and Use of the Microfiltration Apparatus,” the entirety of which is incorporated herein by reference. [Overview of the Initiative] [Means for solving the problem]

[0009] A first aspect of the present invention discloses a microfilter. According to an embodiment, the microfilter includes a polymer layer formed from an epoxy-based photodeterminable material and a plurality of apertures each extending through the polymer layer.

[0010] Another aspect of the present invention discloses a multilayer microfilter. According to an embodiment, the microfilter comprises a first polymer layer formed from an epoxy-based photodestructible material and having a first aperture extending through it, and a second polymer layer formed from an epoxy-based photodestructible material and having a second aperture extending through it, wherein the first and second apertures define at least partially a nonlinear passage extending through the first and second layers.

[0011] A further aspect of the present invention is disclosed: a method for forming a microfilter. According to an embodiment, the method includes: preparing a first layer of photodeterminable material disposed on a substrate; exposing the first layer to energy passing through a mask in order to form a pattern defined by the mask in the first layer of a dried film; forming a polymer layer from the exposed first layer of a dried film having a plurality of apertures extending through the layer, the distribution of which is defined by the pattern; and removing the polymer layer from the substrate.

[0012] A further aspect of the present invention is disclosed: a method for forming a multilayer microfilter. According to an embodiment, the method includes the steps of: forming a first polymer layer containing a plurality of first apertures from a first layer of an epoxy-based photodestructible dry film disposed on a substrate; covering the first polymer layer with a second layer of an epoxy-based photodestructible dry film; and forming a second polymer layer containing a plurality of second apertures from the second layer of the dry film.

[0013] A further aspect of the present invention is disclosed: a filter holder for holding a microfilter. According to the embodiment, the filter holder is designed to keep the filter flat, secure it in place, and allow various analytical steps to be easily performed.

[0014] A further aspect of the present invention is the disclosure of methods for using microfilters. According to embodiments, the method comprises the step of passing a liquid through multiple apertures of a microfilter formed from an epoxy-based photodeterminable dry film, wherein the microfilter has sufficient strength and flexibility to filter the liquid, and the apertures are configured to allow the passage of a first type of body fluid cells but not a second type of body fluid cells. Embodiments include the application of negative pressure using a filter holder or vacuum, which can be connected to a syringe that can be operated manually, semi-manually using a syringe pump, or automatically.

[0015] Another aspect of the present invention is that the filter is coated with an analyte recognition element for capturing target cells from bodily fluids.

[0016] A further aspect of the present invention discloses a method for collecting a liquid sample and transferring the filtered liquid sample using a microfilter inside a filter holder.

[0017] A further aspect of the present invention discloses a method for using a microfilter to perform an analysis. Embodiments provide a method for separating cells, a method for recovering cells from a microfilter by backwashing using a filter holder, and a method for performing an analysis within the filter holder.

[0018] Another aspect of the present invention is the disclosure of analysis for medical applications. [Brief explanation of the drawing]

[0019] A more complete understanding of the present invention and its many associated advantages can easily be obtained by referring to the following detailed description, when considered in relation to the accompanying drawings.

[0020] [Figure 1A] This is a cross-sectional view showing multiple steps in the manufacturing process of a microfilter according to an embodiment of the present invention. [Figure 1B] This is a cross-sectional view showing multiple steps in the manufacturing process of a microfilter according to an embodiment of the present invention. [Figure 1C] This is a cross-sectional view showing multiple steps in the manufacturing process of a microfilter according to an embodiment of the present invention. [Figure 1D] This is a cross-sectional view showing multiple steps in the manufacturing process of a microfilter according to an embodiment of the present invention. [Figure 1E] This is a cross-sectional view showing multiple steps in the manufacturing process of a microfilter according to an embodiment of the present invention. [Figure 2A]According to an exemplary embodiment of the present invention, it is a flowchart showing the manufacturing process of a microfilter. [Figure 2B] According to an embodiment of the present invention, in the process shown in FIG. 2A, it is a flowchart showing the process of forming a microfilter from an exposed dry film. [Figure 3A] According to an exemplary embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of the microfilter 120. [Figure 3B] According to an exemplary embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of the microfilter 120. [Figure 3C] According to an exemplary embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of the microfilter 120. [Figure 3D] According to an exemplary embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of the microfilter 120. [Figure 3E] According to an exemplary embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of the microfilter 120. [Figure 4A] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of a microfilter. [Figure 4B] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of a microfilter. [Figure 4C] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of a microfilter. [Figure 4D] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the manufacturing process of a microfilter. [Figure 5A] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the process of manufacturing a plurality of microfilters from a plurality of layers of an epoxy-based photo-definable dry film. [Figure 5B] According to an embodiment of the present invention, it is a cross-sectional view showing a plurality of stages in the process of manufacturing a plurality of microfilters from a plurality of layers of an epoxy-based photo-definable dry film. [Figure 5C] This is a cross-sectional view showing multiple steps in a process for manufacturing multiple microfilters from multiple layers of epoxy-based photodeficient dry films, according to an embodiment of the present invention. [Figure 5D] This is a cross-sectional view showing multiple steps in a process for manufacturing multiple microfilters from multiple layers of epoxy-based photodeficient dry films, according to an embodiment of the present invention. [Figure 6A] This is a cross-sectional view showing multiple steps in a process for attaching a dry film structure to a support using an electrostatic chuck device, in a microfilter formation process according to an embodiment of the present invention. [Figure 6B] This is a cross-sectional view showing multiple steps in a process for attaching a dry film structure to a support using an electrostatic chuck device, in a microfilter formation process according to an embodiment of the present invention. [Figure 7A] This is a cross-sectional view showing multiple steps in a process for manufacturing a microfilter from a roll of dried film structure according to an embodiment of the present invention. [Figure 7B] This is a cross-sectional view showing multiple steps in a process for manufacturing a microfilter from a roll of dried film structure according to an embodiment of the present invention. [Figure 8A] This is a cross-sectional view showing multiple steps in a process for manufacturing a microfilter from multiple rolls of a dry film structure according to an embodiment of the present invention. [Figure 8B] This is a cross-sectional view showing multiple steps in a process for manufacturing a microfilter from multiple rolls of a dry film structure according to an embodiment of the present invention. [Figure 9A] This is a partial plan view showing the aperture distribution of various microfilters according to an embodiment of the present invention. [Figure 9B] This is a partial plan view showing the aperture distribution of various microfilters according to an embodiment of the present invention. [Figure 9C] This is a partial plan view showing the aperture distribution of various microfilters according to an embodiment of the present invention. [Figure 9D]This is a partial plan view showing the aperture distribution of various microfilters according to an embodiment of the present invention. [Figure 10A] This is a cross-sectional view showing a microfilter according to an embodiment of the present invention, which has apertures of various thicknesses and various shapes, sizes, and distributions. [Figure 10B] This is a cross-sectional view showing a microfilter according to an embodiment of the present invention, which has apertures of various thicknesses and various shapes, sizes, and distributions. [Figure 10C] This is a cross-sectional view showing a microfilter according to an embodiment of the present invention, which has apertures of various thicknesses and various shapes, sizes, and distributions. [Figure 10D] This is a cross-sectional view showing a microfilter according to an embodiment of the present invention, which has apertures of various thicknesses and various shapes, sizes, and distributions. [Figure 10E] This is a cross-sectional view showing a microfilter according to an embodiment of the present invention, which has apertures of various thicknesses and various shapes, sizes, and distributions. [Figure 11] This is a flowchart showing the manufacturing process 1300 of a multilayer microfilter according to an embodiment of the present invention. [Figure 12A] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12B] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12C] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12D] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12E] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12F]This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12G] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12H] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12I] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12J] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12K] This is a cross-sectional view showing multiple steps in the manufacturing process of a multilayer microfilter according to an embodiment of the present invention. [Figure 12L] This is a plan view of a multilayer microfilter according to an embodiment of the present invention. [Figure 13A] This is a plan view showing multiple stages in the process shown in Figure 11. [Figure 13B] This is a plan view showing multiple stages in the process shown in Figure 11. [Figure 14A] This is a cross-sectional view of a multilayer microfilter 1420 according to an embodiment of the present invention. [Figure 14B] This is a cross-sectional view of a multilayer microfilter 1420 according to an embodiment of the present invention. [Figure 14C] Figures 14A and 14B are plan views of the multilayer microfilter 1420. [Figure 15] This is a cross-sectional view of a microfiltration structure including a microfilter and a support structure according to an embodiment of the present invention. [Figure 16A] This is a cross-sectional view of a microfiltration structure, including pores in a microfilter and posts on the microfilter, according to an embodiment of the present invention. [Figure 16B] This is a plan view of a precision filtration structure, including the holes of a microfilter and posts on the microfilter, according to an embodiment of the present invention. [Figure 16C] This is a plan view of a precision filtration structure, including rectangular holes in a microfilter and posts on the microfilter, according to an embodiment of the present invention. [Figure 16D] This is a side view of another microfilter made of two layers of material with different patterns in each layer, according to an embodiment of the present invention. [Figure 16E] This is a side view of another microfilter made of two layers of material with different patterns in each layer, according to an embodiment of the present invention. [Figure 16F] This is a plan view of a microfilter made of two layers of material with different patterns in each layer, according to an embodiment of the present invention. [Figure 16G] This is a side view of another microfilter made of two layers of material with different patterns in each layer, according to an embodiment of the present invention. [Figure 16H] This is a side view of another microfilter made of two layers of material with different patterns in each layer, according to an embodiment of the present invention. [Figure 16I] This is a side view of an apparatus having many wells suitable for many analyses, comprising two layers of material having a solid bottom layer and an upper layer with openings. [Figure 16J] This is a side view of an apparatus having many wells suitable for many analyses, comprising two layers of material having a solid bottom layer and an upper layer with openings. [Figure 17] This is a cross-sectional view of a coated planar microfilter according to an embodiment of the present invention. [Figure 18] This is a cross-sectional view of a coated microfilter structure according to an embodiment of the present invention. [Figure 19] This figure illustrates an example of a microfilter manufactured by lithography using an epoxy-based photodestructible dry film having pores of a position and shape defined by an optical mask, according to an embodiment of the present invention. [Figure 20] This figure shows the components of a filter holder according to an embodiment of the present invention. [Figure 21] This figure shows the assembly process of a microfilter in a filter holder, as shown in Figure 20A-20D, according to an embodiment of the present invention. [Figure 22A] This figure shows an example of an input container for a sample and an input liquid sample, which are mounted on the assembled sample holder shown in Figure 21E, according to an embodiment of the present invention. [Figure 22B] This figure shows an example of an input container for a sample and an input liquid sample, which are mounted on the assembled sample holder shown in Figure 21E, according to an embodiment of the present invention. [Figure 23] This figure shows the formation of an input container attached to the assembled sample holder shown in Figure 21E, using a syringe according to an embodiment of the present invention, without a plunger and adapter, and with a Luer lock. [Figure 24] This figure shows a filtration system for performing analysis, in which a microfilter is incorporated inside a filter holder, according to an embodiment of the present invention. [Figure 25] This figure shows a filtration system for performing several steps of analysis together with an input container to be removed, according to an embodiment of the present invention. [Figure 26] This figure shows a filtration system using the filtration system shown in Figure 24, according to an embodiment of the present invention, in which the syringe outlet is drawn by a syringe pump. [Figure 27A] This embodiment of the present invention shows a configuration in which a vacuum container holder draws a liquid sample from an input sample holder via a filter into the vacuum container. [Figure 27B] This embodiment of the present invention shows a configuration in which a vacuum container holder draws a liquid sample from an input sample holder via a filter into the vacuum container. [Figure 28A] This figure shows a configuration according to an embodiment of the present invention, in which a vacutainer holder is used to draw a liquid sample from a patient through a filter into a vacutainer. This can be used to filter circulating tumor cells during blood collection according to an embodiment of the present invention. [Figure 28B] This figure shows a configuration according to an embodiment of the present invention, in which a vacutainer holder is used to draw a liquid sample from a patient through a filter into a vacutainer. This can be used to filter circulating tumor cells during blood collection according to an embodiment of the present invention. [Figure 29] This flowchart shows options for a procedure to collect and test a liquid sample according to an embodiment of the present invention, and an option to filter out rare cells when blood is collected using a vacuum container. [Figure 30A] This is a flowchart showing a filtration process using a microfilter according to an embodiment of the present invention. [Figure 30B] This is a flowchart showing a filtration process using a microfilter according to an embodiment of the present invention. [Figure 30C] This flowchart shows a process for performing several types of analysis of cells collected on a microfilter using a filter holder, according to an embodiment of the present invention. [Modes for carrying out the invention]

[0021] By referring to the drawings presented here, embodiments of the present invention are disclosed schematically and in detail, such that similar reference numerals designate the same or corresponding parts throughout several figures.

[0022] The details defined in the description of the configuration and elements are provided solely to aid in a comprehensive understanding of the present invention. Those skilled in the art will recognize that various modifications and variations of the embodiments described herein can be made without departing from the scope and spirit of the invention. Furthermore, well-known functions or structures have been omitted for clarity and brevity. Several exemplary embodiments of the present invention are described below in the context of commercial use. Such exemplary implementations do not limit the scope of the invention as defined in the appended claims.

[0023] Aspects of the present invention generally relate to a microfilter comprising a polymer layer formed from an epoxy-based photodestructible dry film. The microfilter includes a plurality of apertures extending through the polymer layer, each penetrating the layer. In some embodiments, the microfilter may be formed by exposing the dry film with energy passing through a mask and developing the exposed dry film. In some embodiments, the dry film may be exposed with energy in the form of ultraviolet (UV) light. In other embodiments, the dry film may be exposed with energy in the form of X-rays. In some embodiments, the polymer layer has sufficient strength and flexibility to filter liquids. In some embodiments, the apertures are formed to be sized to allow passage through a first type of fluid cell but not through a second type of fluid cell.

[0024] In detail, in some embodiments, a microfilter may be used to perform an assay on body fluids. In some embodiments, a microfilter may be used to separate and detect rare cells from body fluids. In certain embodiments, a microfilter may be used to collect circulating tumor cells (CTCs) from peripheral blood of cancer patients that has passed through the microfilter. In certain embodiments, a microfilter can be used to collect circulating endothelial cells, fetal cells, and other large cells from blood and body fluids. In certain embodiments, a microfilter may be used to collect large cells from processed tissue samples, such as bone marrow. In some embodiments, cells collected using a microfilter may be used in downstream processes such as cell identification, counting, characterization, and culture.

[0025] More specifically, in certain embodiments, multiple layers of an epoxy-based photodestructible dry film may be simultaneously exposed by energy for large-scale production of microfilters. In some embodiments, a laminate of epoxy-based photodestructible dry film layers is formed, and all of the dry film layers of the laminate are simultaneously exposed by energy. In some embodiments, a dry film structure including an epoxy-based photodestructible dry film formed on a substrate is prepared in the form of a roll. In such embodiments, a portion of the structure may be unfolded from the roll to expose the dry film by energy. In some embodiments, multiple portions of multiple rolls may be simultaneously exposed by energy.

[0026] Figures 1A to 1E are cross-sectional views showing multiple steps in the manufacturing process of a microfilter 120 according to an embodiment of the present invention. Figure 2A is a flowchart showing the manufacturing process 200 of a microfilter according to an embodiment of the present invention. Hereinafter, the exemplary process shown in Figure 2A will be described with reference to Figures 1A to 1E. Furthermore, other embodiments of the process shown in Figure 2A will be described later with reference to Figures 3A to 8B.

[0027] In block 220 of Figure 2A, a layer of photodestructible epoxy-based dry film 100 (sometimes referred to herein as “dry film 100”) is prepared on the substrate 180. In some embodiments, in block 220, the dry film 100 is laminated onto the substrate 180. In one embodiment, in block 220, a thin layer of metallic material, such as copper, is coated onto a silicon wafer, and the dry film 100 is laminated onto the metallic material. In other embodiments, in block 220, a dry film 100 already attached to the substrate 180 may be acquired and prepared. In this specification, “photodestructible epoxy-based” material means a material including or formed from a photodestructible epoxy resin such as a multifunctional epoxy resin, a bisphenol A epoxy resin, or an epoxidized polyfunctional bisphenol A formaldehyde novolac resin. Examples of photo-blocizable epoxy resins are shown in U.S. Patent Nos. 7,449,280, 6,716,568, 6,391,523, and 6,558,868, and the entire contents of those documents are incorporated herein by reference. Examples of photo-blocizable epoxy resins are also shown in U.S. Patent Publication Nos. 2010 / 0068648 and 2010 / 0068649, and the entire contents of those documents are incorporated herein by reference. In this specification, “epoxy-based photo-blocizable dry film” means a dry film containing or formed from an epoxy-based photo-blocizable substance. U.S. Patent Nos. 7,449,280, 6,391,523, and 6,558,868, as well as U.S. Patent Publication Nos. 2010 / 0068648 and 2010 / 0068649, provide examples of photo-blocizable epoxy films that may be used in embodiments of the present invention. Photo-blocizable epoxy films are not limited to those described herein.

[0028] A liquid resist foam of an epoxy-based photo-definable dry film, before being applied to a substrate, can be spin-coated onto the substrate and dried to obtain a dry film on the substrate.

[0029] In certain embodiments, the substrate 180 is a thin copper foil. In some embodiments, the substrate is preferably smooth because any irregularities on the substrate surface to which the dried film is applied will be transferred to the surface of the dried film. In some embodiments, a thin copper film is preferred as the substrate so that the substrate can be removed in a relatively short time. In other embodiments, the substrate 180 may be a silicon wafer, a polyimide film such as Kapton, or any other suitable material.

[0030] As shown in Figures 1B and 1C, in block 240, the dry film 100 is exposed by energy through the mask 199 to form the exposed dry film 110. In the embodiments shown in Figures 1B and 1C, the dried film 100 is exposed by the energy of ultraviolet (UV) light passing through an optical mask 199 having a mask portion 197 that is transparent to UV light and a mask pattern 198 formed of a thin film of a material that is opaque to UV light. In another embodiment, the dried film 100 in block 240 may be exposed by X-rays passing through an X-ray mask instead of being exposed by UV light passing through the optical mask 199.

[0031] In the embodiments shown in Figures 1A to 1E, the photo-blocatable epoxy-based dry film 100 is a negative-type resist. "Negative-type resist" as used herein refers to a photo-blocatable substance that polymerizes when exposed to certain energies, such as UV light or X-rays. Examples of photo-blocatable dry films of negative-type resist epoxy systems, which may be used in embodiments of the present invention, can be used according to embodiments of the present invention.

[0032] As shown in Figure 1C, the portion of the exposed dried film 110 exposed to UV light through the mask 199 becomes polymerized, while portion 116 remains non-polymerized. The polymerized and non-polymerized portions of the exposed dried film 100 form a pattern 118 defined by the optical mask 199. In particular, the pattern 118 of the exposed dried film 110 is defined by the pattern 198 of the optical mask 199, and the pattern 198 is formed of a material opaque to UV light. In one embodiment, the pattern 198 may be formed of a thin film of a material opaque to UV light, such as a chromium thin film.

[0033] In another embodiment, a positive epoxy-based photoselectable dry film may be used instead of a negative dry film. In such embodiments, the process for forming a microfilter from a positive dry film is similar to the process for forming a microfilter described in relation to Figures 1A to 2A, except that a different mask may be used, as described below in relation to Figures 4A to 4D. In this specification, “positive resist” refers to a photoselectable material whose polymerization bonds are broken when exposed to certain energies, such as UV light or X-rays. In some embodiments, the positive resist may be a polydimethylglutarimide (such as PMGI, LOR, available from MicroChem), an acetate and xylene-free resist (such as the S1800® series resists available from Shipley Corp.), or a resist based on another type of positive resist. For resist layers thicker than a few microns, negative resists are generally more sensitive than positive resists. Most polymer resists belong to the category of positive resist films. Examples of dry film positive resists that may be used include polymethyl methacrylate (PMMA) and synthetic polymers of methyl methacrylate. Other examples of positive resists include acrylics, polyimides, and polyesters such as polyethylene terephthalate (PET) (MYLAR®). In some embodiments, a microfilter may be formed from a non-epoxy photo-blocatable dry film according to embodiments of the present invention. In such embodiments, the dry film may be a positive or negative resist. In other embodiments, the microfilter may be formed from a photo-blocatable liquid instead of a dry film. In such embodiments, the photo-blocatable liquid may be a positive or negative resist. In some embodiments, the photo-blocatable liquid may be a liquid polyimide. In such embodiments, the photo-blocatable liquid polyimide may be a positive or negative resist. The liquid resist is spin-coated onto a substrate and dried to form a dry film on the substrate.

[0034] In block 260, a microfilter 120 having a plurality of apertures 122 extending through the microfilter is formed from an exposed dried film 110. In one embodiment, the microfilter 120 includes a polymer layer formed from an epoxy-based photodeterminable dried film and a plurality of apertures extending through the polymer layer. In each of the embodiments of the present invention described herein, the microfilter includes one or more polymer layers and one or more apertures extending through each of the one or more polymer layers. In this specification, “aperture” means any type of passage, hole, trench, gap, hole, etc., extending between layers or the outer surfaces of other structures. In the embodiments shown in Figures 1A to 1E, the aperture 122 is a hole 122.

[0035] In the embodiments shown in Figures 1A to 1E, the exposed dried film 110 is developed to remove the non-polymerized portion 116 and form a microfilter 120 having pores 122. In one embodiment, the exposed and dried film 110 is developed by applying a developer to the dried film 100 to dissolve the non-polymerized portions 116. In some embodiments, the developer is an aqueous solution that dissolves the non-polymerized portions 116 when the exposed and dried film 110 is immersed in the developer. In block 280, the microfilter 120 having pores 122 is removed from the substrate 180 to form a self-supporting microfilter 120, as shown in Figure 1E. In some embodiments, the microfilter is a structure of one or more polymer layers including one or more apertures extending between the outer surfaces of the structure, the structure having sufficient strength and flexibility to filter a liquid passing through one or more apertures. In some embodiments, the microfilter may include apertures that are so small in size that one or more types of body fluid cells cannot pass through the apertures when a body fluid or a liquid containing body fluids passes through the filter, and the dimensions of the apertures are also so small that one or more other types of body fluid cells cannot pass through the filter. In this specification, “body fluid cells” refers to any cells found in a patient’s body fluids, including red blood cells, white blood cells, or large, rare cells such as CTCs and fetal cells, circulating endothelial cells, etc. One way to obtain tumor cells is to remove bone fragments and bring the bone into a liquid state. In some embodiments, the microfilter includes an aperture sized to allow a large number of red blood cells to pass through but not a large number of CTCs. In some embodiments, the microfilter, formed from one or more layers of an epoxy-based photodestructible dry film, may be a polymer microfilter.

[0036] The following describes various embodiments of the process shown in Figure 2A in relation to Figures 2B to 8B. As described above, in one embodiment, the substrate 180 may be copper foil. In such an embodiment, as one variation of block 280, the copper substrate 180 may be removed from the microfilter 120 using nitric acid, iron chloride, or another known reagent. In one embodiment, the reagent may be used to etch off the copper substrate 180 for the purpose of removing the copper substrate 180 from the microfilter 120. In another embodiment, the substrate 180 may be another type of metal foil, such as aluminum, which may be removed in block 280 by a known method.

[0037] Figure 2B is a flowchart showing the process of forming a microfilter from an exposed and dried film in block 260 shown in Figure 2A, according to an embodiment of the present invention. In one embodiment, the microfilter formation process 260 includes the step of forming a polymer layer containing a plurality of apertures from the exposed and dried film. In the embodiment of Figure 2B, a post-bake process is performed on the exposed and dried film 110 placed on the substrate 180 in block 262. In one embodiment, the post-bake process includes the step of exposing the dried film 110 to a relatively high temperature in order to post-bake the dried film 110. In block 264, the dried film 110 is developed by applying a developer to the dried film 110 as described above for Figures 1A to 1E. In block 266, a hard-bake process is performed on the developed and dried film 110. In one embodiment, the hard-bake process includes the step of exposing the dried film 110 to a relatively high temperature. In some embodiments of the process shown in Figure 2B, the hard-bake process in block 266 may be omitted. In such embodiments, the microfilter 120 is formed by post-baking the exposed dried film 110 in block 262 and developing the dried film 110 in block 264. The process described above in relation to Figure 2B can be used for any embodiment described herein. Furthermore, in any embodiment of the present invention described herein, the process for forming the polymer layer of the microfilter from an epoxy-based photodeterminable dried film may include, as described above, the steps of exposing the dried film with energy, performing a post-bake process, developing the exposed dried film, and / or post-baking the exposed dried film.

[0038] Figures 3A to 3E are cross-sectional views showing several steps in the manufacturing process of a microfilter 120 according to an embodiment of the present invention. In the embodiments shown in Figures 3A to 3E, the substrate is a polyimide film 181. In block 220, a layer of epoxy-based photodeterminable dry film 100 disposed on the polyimide film 181 is provided with a separator 182 positioned between a portion of the dry film 100 and the polyimide film 181. In the embodiment shown in Figure 3A, the separator 182 is formed along the edge of the dry film 100 between a portion of the dry film 100 and the polyimide film 181. In some embodiments, the separator 182 may be provided along one or more edges of the dry film 100, or at other locations between the dry film 100 and the polyimide film 181. The separator 182 may be formed from a polyimide film (such as a KAPTON film) or from any other suitable material that can be laminated on the dry film 100 and withstand the temperatures of the hard bake process.

[0039] As shown in Figures 3B to 3C, and as described above in relation to Figures 1B to 1D, in blocks 240 and 260, the dried film 100 is exposed by energy and a microfilter 120 is formed from the exposed dried film 110. In one embodiment, the microfilter 120 includes a polymer layer through which multiple apertures penetrate. In the embodiments shown in Figures 3A to 3E, the microfilter 120 is removed from the polyimide film 181 by grasping the exposed end of the separator 182 and using the separator 182 to peel the microfilter 120 from the polyimide film 181. After removing the dried film 100 from the polyimide layer 181 as shown in Figure 3D, the separator 182 is removed from the dried film 100 as shown in Figure 3E to obtain a self-supporting microfilter 120. The steps of removing the microfilter 120 from layer 181 and removing the separator 182 from the microfilter 120 are two steps performed in one modification of block 280 according to an embodiment of the present invention.

[0040] In another embodiment of the process shown in Figure 2A, a liquid resist may be used instead of the dried film 100. In such an embodiment, as one variation of block 210, the substrate is coated with a thin layer of metallic material, and an epoxy-based liquid photoresist is spin-coated onto the metallic material to prepare a layer of epoxy-based photo-deficient material on the substrate. In some embodiments, the substrate may be a silicon wafer, the metallic material may be copper, and the liquid photoresist may be an epoxy-based photo-deficient liquid. In some embodiments, the epoxy-based photo-deficient liquid is a liquid negative resist such as SU-8. As described above with respect to Figures 1B to 1D, in blocks 240 and 260, the layer of epoxy-based photo-deficient material is exposed by energy, and a microfilter 120 is formed from the exposed layer. As described above, as one variation of block 280, the microfilter 120 is peeled off from the substrate by etching off the metallic material using a conventional process. In some embodiments, the liquid resist may be a liquid negative resist such as SU-8 or KMPR®, available from MicroChem Corp.

[0041] In other alternative embodiments of the process shown in Figure 2A, a liquid negative resist may be used instead of the dried film 100, and a positive resist may be used between the negative resist and the substrate as a release layer. In such embodiments, as one variation of block 210, a liquid positive resist is spin-coated onto the substrate to form a layer of epoxy-based photodestructible material on the substrate, the positive resist is exposed with an appropriate amount of irradiation energy (such as UV light) depending on the thickness of the coating, and then a liquid epoxy-based negative resist is spin-coated onto the positive resist. In some embodiments, the positive resist may be exposed by energy without the use of a mask. The layer of epoxy-based photodestructible material is exposed by energy, and the microfilter 120 is formed from the exposed layer in blocks 240 and 260, as described above for Figures 1B to 1D. In such embodiments, as one variation of block 280, the microfilter 120 is peeled off from the substrate by developing the positive resist. In one embodiment, the same developer may be used to develop both positive and negative resists. In another embodiment, one developer may be used to form pores in the microfilter 120, and another developer may be used to peel the microfilter from the substrate. In yet another embodiment, a dry film positive resist may be used as the release layer instead of a liquid positive resist. Examples of dry film positive resists that may be used include polymethyl methacrylate (PMMA) and synthetic polymers of methyl methacrylate.

[0042] In other embodiments, a photodeterminable dry film 100 of a negative epoxy system may be used in combination with the positive resist release layer. Such embodiments are similar to the embodiments using the positive resist release layer described above, except that instead of spin-coating with a liquid negative resist in block 210, a layer of the negative dry film 100 may be applied on top of a spin-coated positive resist.

[0043] Figures 4A to 4D are cross-sectional views showing multiple steps in the manufacturing process of a microfilter 420 according to an embodiment of the present invention. In the embodiments shown in Figures 4A to 4D, a layer of a photodeterminable dry film 400 (sometimes referred to herein as "dry film 400") of a positive epoxy system is prepared in block 220 on a substrate 180. In one embodiment, the positive dry film 400 is placed on the substrate 180 in block 220. As shown in Figure 4B, as described above for Figures 1B and 1C, the positive dry film 400 is exposed by energy in block 240. However, the difference is that the exposed portion of the dry film 400 does not become polymerized, but rather the polymerization bonds of the dry film 400 are broken in the portion 416 exposed by energy (e.g., UV light) that has passed through the mask 499. A pattern 418, consisting of exposed portions 416 and unexposed portions, is formed on the exposed dry film 410. As shown in Figure 4B, the mask 499 includes a transparent portion 497 and an opaque portion 498. As described above, the mask 199 in Figure 1B is used with a negative-type resist and is configured to cover the portion of the dried film 100 where pores will be formed. In the embodiments shown in Figures 4A to 4D, the opaque portion 498 of the mask 499 is configured to cover all portions of the positive-type dried film 400 except where apertures will be formed, thereby allowing UV light to pass through the mask 499 to the portion of the positive-type dried film 400 where apertures are to be formed.

[0044] As one variation of block 260 in the embodiments shown in Figures 4A to 4D, a microfilter 420 is formed from the exposed dried film 410 by developing the dried film 410 using a developer that dissolves a portion 416 of the dried film 400 in which the polymerization bonds have been broken. As shown in Figure 4D, in block 280, a microfilter 420 having apertures 422 is removed from the substrate 180 to form an independent polymer microfilter 420. In some embodiments, the microfilter 420 includes a polymer layer formed from an epoxy-based photodeterminable dry film and includes a plurality of apertures extending through the polymer layer. In some embodiments, the apertures 422 are holes 422. In some embodiments, in block 280, the microfilter 420 may be peeled off from the substrate by developing a positive resist as described above.

[0045] Figures 5A to 5D are cross-sectional views showing multiple steps in a process for manufacturing multiple microfilters from multiple layers of epoxy-based photodeficient dry films according to embodiments of the present invention. In one embodiment of the process shown in Figure 2A, multiple dry film structures 501 are prepared in block 220, each containing a layer of epoxy-based photodeficient dry film 500 (sometimes referred to herein as "dry film 500") disposed on a substrate 580. In embodiments shown in Figures 5A to 5D, the dry film 500 disposed on the substrate 580 in block 220 is prepared by stacking the structures 501 on a support 590, as shown in Figure 5A.

[0046] In one embodiment, as shown in Figure 5A, multiple dry films 500 contained in the laminate of structure 501 are simultaneously exposed in block 240 of Figure 2A by the energy of X-rays passing through the X-ray mask 599. In some embodiments, X-rays penetrate considerably deeper than UV light. Unlike UV light, X-rays do not diverge to shapes significantly smaller than 1 micron within materials with a thickness of less than 5 mm. In some embodiments, X-ray lithography may typically be performed on a synchrotron beamline. X-ray lithography can also be used for both negative and positive resists. In the embodiments shown in Figures 5A to 5D, the dry films 500 are each negative resists. In other embodiments, the dry films 500 may be positive resists. In such embodiments, a mask configured to form an aperture in the positive resist can be used, similar to what is described above with respect to the mask 499 in Figure 4B. Furthermore, in embodiments where the dried film 500 is a positive-type resist, the dried films 500 can be directly laminated onto each other by being attached to the support 590, rather than each being placed on a separate substrate.

[0047] As shown in Figure 5B, the portion of each dried film 500 exposed to X-rays through the mask 599 becomes polymerized, while the portion 516 of the dried film 500 remains unpolymerized. The polymerized and unpolymerized portions of each dried film 500 form a pattern 518 defined by the pattern 598 of the optical mask 599. In some embodiments, the mask 599 includes an X-ray-transparent portion 597 and a pattern 598 configured to substantially block X-rays. In some embodiments, the pattern 598 is formed of gold. In some embodiments, the X-ray-transparent portion 597 may be a thin graphite sheet or a silicon wafer. In the embodiments shown in Figures 5A to 5D, each substrate 580 transmits most of the X-ray energy incident on it. In some embodiments, the substrate 580 is formed of a metal foil. In such embodiments, if the metal foil is sufficiently thin, each substrate 580 transmits most of the X-ray energy incident on it. In some embodiments, the number of structures 501 that may be stacked and exposed simultaneously is determined based on the attenuation of the X-ray irradiation amount that occurs in the X-rays passing through the metal foil.

[0048] In one embodiment, as a modification of block 260, a plurality of exposed dried films 510 are developed in a manner similar to that described above in relation to Figures 1A to 1E, so as to form a plurality of microfilters 520, each having an aperture 522. In one embodiment, the aperture 522 is a hole 522. In some embodiments, the processes shown in Figures 5B and 5C may be performed in a modification of block 260. In such embodiments, the structures 501 are separated from each other as shown in Figure 5B, and a post-bake process is performed on the exposed dried films 510 placed on each substrate 580 in a modification of block 262. In one embodiment, each of the exposed dried films 510 is developed as described above in a modification of block 264 to form a hole 522 in each of the dried films 500, as shown in Figure 5C. In some embodiments, a hard bake process is performed on the dried films 510 placed on each substrate 580 in a modification of block 266 to form microfilters 520 having holes 522. In other embodiments, the hard bake treatment may be omitted. In one embodiment, in one modification of block 280, as shown in Figure 5, the substrate 580 is chemically removed from the microfilter 520 as described above in order to obtain independent microfilters 520 having pores 522. In one embodiment, each microfilter 520 is a polymer layer containing apertures 522.

[0049] As described above, in one embodiment, each of the substrates 580 may be formed from a metal foil. In another embodiment, the substrate 580 may be a polymer substrate that transmits most of the X-rays applied to the substrate and has a melting point higher than the post-bake temperature of the dry film 500. For example, in one embodiment, the substrate 580 may be formed from a positive resist. In such an embodiment, the substrate 580 may be exposed to energy sufficient to break the polymerization bonds of the positive resist, such as UV light or X-rays, so that the substrate 580 is chemically removed by a developer in block 280 of Figure 2A. In another embodiment, the substrate 580 may be a polyimide film, which may be removed in block 280 by peeling the polyimide substrate 580 from the microfilter 520.

[0050] In another embodiment, multiple layers of an epoxy-based photodeterminable dry film 500 (sometimes referred to herein as “dry film 500”) may be exposed simultaneously without each layer being placed on its respective substrate and laminated. In such an embodiment, the dry films 500 are laminated on a support 590 without substrates placed between adjacent dry films 500 in one variation of block 220. The laminated dry films 500 are exposed in one variation of block 240. In some embodiments, the process shown in Figure 2B may be performed in block 260. In such embodiments, the exposed dry films 510 are separated and placed on individual substrates, and a post-bake process is performed on these exposed dry films 510 on individual substrates in one variation of block 262. In such embodiments, the substrates used are capable of withstanding the post-bake temperature and are dissolvable with water or one or more chemicals. The exposed dry films 510 may be developed in block 264 while attached to their respective substrates and hard-baked in block 266. In block 280, the substrate 580 is removed from the microfilter 520 formed from the exposed dried film 510.

[0051] In one embodiment, the structure 501 may be attached to the support 590 using an adhesive, clamp, or any other suitable mechanism or method. In some embodiments, the structure 501 is held to the support by an electrostatic chuck. Figures 6A and 6B are cross-sectional views showing multiple steps in the process of attaching the dry film structure 501 to the support using an electrostatic chuck device 600 in the process of forming a microfilter according to an embodiment of the present invention. In the embodiments shown in Figures 6A and 6B, multiple dry film structures 501 each include a layer of photodefined epoxy-based dry film 500 disposed on a substrate 580 and are prepared by being laminated on a support 690 as shown in Figure 6A. As shown in Figure 6A, the support 690 includes a water-cooled frame 692 having a duct 693, an insulator 664 disposed on the frame 692, and a conductive layer 662 disposed on the insulator 664. Furthermore, as shown in Figure 6A, a transparent conductive layer 660 is placed on a laminate of structure 501, and the laminate of structure 501 is placed between the conductive layer 660 and 662. Also, as shown in Figure 6A, the circuit connecting the conductive layers is open, and the voltage 665 applied to the conductive layer is zero.

[0052] As shown in Figure 6B, when the circuit between the conductive layers is closed and a non-zero voltage 665 is applied between the conductive layers 660 and 662, the apparatus 600 presses the structure 501 between the conductive layers 660 and 662. With the structure 501 pressed by the apparatus 600, the dried film 500 may be irradiated with X-rays through the X-ray mask 599 as described above in relation to Figures 5A to 5D. In one embodiment, stacking the structure 501 on the apparatus 600 and pressing the structure 501 together using the apparatus 600 may be performed in one modification of block 220 in Figure 2A, as described above in relation to Figures 6A and 6B. The use of an electrostatic chuck apparatus has been described above in relation to forming a microfilter from dried films placed on each substrate, but in another embodiment, an electrostatic chuck may be similarly used to press a stack of freestanding polymer films, such as freestanding dried films not placed on each substrate. Such multiple independent dried films may be stacked and pressed together during the process of forming multiple microfilters from the multiple dried films.

[0053] Figures 7A and 7B are cross-sectional views showing several steps in a process for manufacturing a microfilter from a roll of a dry film structure according to an embodiment of the present invention. In the embodiments shown in Figures 7A and 7B, the dry film structure 785 is prepared in the form of a roll 702 of the dry film structure 785. The dry film structure 785 includes a layer of an epoxy-based photodeterminable dry film 700 (sometimes referred to herein as “dry film 700”) disposed on a removable substrate 782. In some embodiments, the substrate 782 may be a chemically dissolvable metal foil. In some embodiments, the metal foil may contain aluminum or copper and may be etched off as described above. In the embodiments shown in Figures 7A and 7B, one end of the roll 702 is positioned on roller 774 and the other end on roller 775. The working portion 787 of the dry film structure 785 is stretched between rollers 774 and 775, held substantially flat by roller 770, and exposed by energy passing through a mask 199.

[0054] In one embodiment of the process shown in Figure 2A, in one modification of block 220, a portion of the dry film structure 785 is unrolled from the roll 702 and the portion of the structure 785 is advanced in the direction of arrow 772, thereby providing a working portion 787 of the structure 785 between the support 791 and the mask 199, thereby preparing a layer of photo-defined epoxy-based dry film 700 on the substrate 782. In some embodiments, the working portion 787 provided in block 220 includes a portion of the dry film 700 that has not been patterned by exposure of energy through the mask. In some embodiments, the support 791 and the mask 199 are moved away from the structure 785 as the structure 785 is advanced.

[0055] In the embodiments shown in Figures 7A and 7B, in one modification of block 240 of Figure 2A, as shown in Figure 7B, the mask 199 and support 791 are moved to a position adjacent to the structure 785, and the dried film 700 is exposed by energy through the mask 199. In the embodiments shown in Figures 7A and 7B, the mask 199 is an optical mask and the energy is UV light, but as described above, different types of energy may be used with different masks. In the embodiments shown in Figures 7A and 7B, the dried film 700 is a negative resist. In other embodiments, the dried film 700 may be a positive resist. In such embodiments, a mask configured to form holes in the positive resist may be used, as described above in relation to the mask 499 of Figure 4B. As described above in relation to other embodiments, when the dried film 700 is exposed by energy through the mask, a pattern is formed on the dried film 700. In one embodiment, the support 791 presses the structure 785, as shown in Figure 7B, stretching the dry film 700 in preparation for the exposure process, thereby providing the dry film 700 with further tension and stability during the exposure process. In some embodiments, after the dry film 700 has been exposed, the structure 785 may be advanced again as described above to prepare a new working portion 787 in block 220 that has not been exposed, and the new working portion 787 may be exposed in block 240 as described above. In some embodiments, this process of advancing the structure 785 to expose the dry film 700 may be repeated continuously. In some embodiments, this process may be repeated until most or all of the dry film 700 has undergone the exposure process.

[0056] In some embodiments, in one modification of block 260, a microfilter having an aperture is formed from the exposed portion of the dried film 700 by developing the exposed portion, as described above in relation to other embodiments. In such embodiments, the exposed portion of the dried film 700 may be developed before being wound onto the roller 775, or after all desired portions of the dried film 700 have been exposed. In some embodiments, the process shown in Figure 2B may be performed in block 260. In such embodiments, the exposed portion of the dried film 700 may advance through a post-bake furnace in block 262, and the exposed portion of the dried film 700 may be developed in block 264 and then hard-bake in block 266. In other embodiments, the processing in blocks 262, 264, and 266 may be performed after all desired portions of the dried film 700 have been exposed. In some embodiments, the hard-bake process may be omitted.

[0057] In some embodiments, after developing the exposed and dried film 700, the substrate 782 is removed in block 280 as described above in relation to other embodiments. In some embodiments, after removing the substrate 782, individual microfilters are cut from the roll of dried film on which the microfilters are formed. In some embodiments, forming microfilters from a roll of dried film prepared as a roll simplifies the manufacture of microfilters according to the embodiments of the present invention and may enable automation of the manufacturing process.

[0058] Figures 8A and 8B are cross-sectional views showing several steps in a process for manufacturing a microfilter from multiple rolls of a dry film structure according to an embodiment of the present invention. The embodiment shown in Figures 8A and 8B is similar to the embodiment shown in Figures 7A and 7B, except that the layers of epoxy-based photodeficient dry film 700 on multiple rolls 702 are simultaneously exposed by energy. In such an embodiment, in one modification of block 220, multiple layers of epoxy-based photodeficient dry film 700, each disposed on a substrate 782, are prepared by advancing the structure 785 of each roll 702 in the direction of arrow 772 to supply a laminate 887 of the structure 785 between the support 891 and the mask 899. In the embodiments shown in Figures 8A and 8B, the mask 899 and support 891 are moved to a position adjacent to the laminate 887, and the portion of the dry film 700 of the laminate 887 positioned between the mask 899 and support 891 is simultaneously exposed by energy through the mask 899, as shown in Figure 8B, in one modification of block 240 in Figure 2A. In the embodiments shown in Figures 8A and 8B, each of the dry films 700 is a negative resist. In other embodiments, the dry film 700 may be a positive resist. In such embodiments, a mask configured to form an aperture in the positive resist may be used, as described above in relation to the mask 499 in Figure 4B. Further processes for forming a microfilter from the dry film 700 are similar to the processes described above in relation to the embodiments shown in Figures 7A and 7B.

[0059] In the embodiments shown in Figures 8A and 8B, the support 891 is positioned on a water-cooled frame 692 including a duct 693. Also, as shown in Figure 8B, the working portion 887 of the structure 785 may be securely held in place between the support 891 and the mask 899 by a clamp 860. In another embodiment, the laminate 887 may be securely held using an electrostatic chuck, as described above in relation to other embodiments. In some embodiments, the number of dry films 700 to be exposed simultaneously may be determined based on the accuracy required when exposing a laminate of a certain number of films. In some embodiments, the manufacture of the microfilter may be simplified and / or facilitate the mass production of the microfilter by forming a microfilter from multiple dry films prepared as multiple rolls as described above.

[0060] In embodiments where a non-epoxy drying film is used, the drying film may be prepared in the form of a roll without a substrate. In such embodiments, each roll 702 contains only the drying film and no substrate. In embodiments where an epoxy drying film is used, each roll 702 may include an additional coating layer on the drying film 700. In such embodiments, the substrate 782 is positioned on the first side of the drying film 700, and the coating layer is positioned on the opposite side of the drying film 700. In some embodiments, microfabrication based on lithography according to embodiments of the present invention may enable efficient mass production of microfilters with highly uniform precision. In some embodiments, by fabricating microfilters according to embodiments of the present invention, the porosity and pore uniformity of the produced microfilters may be increased.

[0061] Figures 9A to 9D are partial plan views showing the aperture distribution of various microfilters according to embodiments of the present invention. In some embodiments, microfilters with apertures having different sizes, shapes, and distributions may be fabricated. In some embodiments, for specific applications of the microfilter, a certain combination of aperture size, shape, and distribution may be more advantageous than other combinations. For example, in some embodiments, a microfilter having circular pores, each with a diameter of 7-8 microns, may be preferred for the precision filtering of rare cells such as circulating tumor cells or fetal cells in the blood. In some applications, a microfilter having circular pores, each with a diameter of 7-8 microns, can capture rare cells while retaining a very small proportion of blood cells.

[0062] In the embodiments shown in Figures 9A and 9B, microfilters 910 and 912 have uniformly distributed pores 920 and 922, respectively. The pores 920, like the pores 922, are uniform in size. In the embodiment shown in Figure 9C, microfilter 914 includes uniform pores 924, which are distributed within the microfilter 914 in several groups. In the embodiment shown in Figure 9D, microfilter 916 includes a plurality of pores 926 of a first size and a plurality of pores 928 of a second size. In other embodiments, any of the pores 920, 922, 924, 926, and 928 may be any other type of aperture. Any of the microfilters 910, 912, 914, and 916 may be manufactured using any of the microfilter manufacturing processes described above according to embodiments of the present invention. Furthermore, any of the microfilter manufacturing processes described above according to embodiments of the present invention may be used to form apertures of multiple different cross-sectional shapes. For example, in one embodiment, an aperture having a cross-sectional shape such as a circle, triangle, square, rectangle, oval, ellipse, trapezoid, or parallelogram may be formed.

[0063] Microfilters having varying thicknesses and apertures of varying shapes, sizes, and distributions may be manufactured by embodiments of the present invention described herein. Figures 10A to 10D are cross-sectional views of microfilters having varying thicknesses and apertures of varying shapes, sizes, and distributions according to embodiments of the present invention. Figures 10A and 10B show microfilters 1010 and 1012 formed by one of the processes described above according to embodiments of the present invention, respectively. Microfilter 1010 includes a plurality of holes 1020, each having a width of 1040. The microfilter has a thickness 1030 that is substantially perpendicular to the width 1040 of the holes 1020. In the embodiment shown in Figure 10A, the thickness 1030 is not significantly greater than the width 1040. In some embodiments, the thickness of the microfilter is preferably approximately the same as the width of one or more of the holes in the microfilter to reduce the pressure required for the sample to pass through the holes. In some applications, if the thickness of the microfilter is significantly greater than the width of some or all of the holes, it may require much greater pressure on the microfilter to pass the sample through compared to when the thickness of the microfilter is approximately the same as the width of some or all of the holes. Forcing a sample through a filter with relatively high pressure can risk deforming the shape of one or more holes or damaging the microfilter.

[0064] For example, in one embodiment, a microfilter with a thickness of 8 to 14 microns may be preferred for the precision filtration of rare cells such as CTCs or fetal cells in the blood. In one embodiment, a microfilter for such an application may have pores with a diameter of 7 to 8 microns and a thickness of 8 to 14 microns. In another embodiment, a microfilter for such an application may include a rectangular aperture having a width of 5 to 7 microns and a length greater than 7 microns, where both the length and width of the aperture are measured approximately perpendicular to the thickness of the microfilter. In one embodiment, the rectangular aperture may be an elongated trench. In one embodiment, the width of the aperture provided in the microfilter may preferably be close in size to the thickness of the microfilter. In some embodiments, the thickness of the microfilter is less than 10 times the width of some or all of the pores. In other embodiments, the thickness of the microfilter is within the range of 10 microns, which is the width of some or all of the pores. Microfilters formed according to embodiments of the present invention may be used in applications other than capturing circulating tumor cells from blood. In some embodiments, the desired aperture geometry, aperture dimensions, aperture distribution, microfilter material, microfilter thickness, microfilter size, etc., may vary depending on the application. In some embodiments, the desired aperture geometry, dimensions, and distribution can be achieved by using a suitable mask, such as an optical mask or X-ray mask. In some embodiments, the material strength of the microfilter may be important to prevent damage to the filter material or deformation of the aperture shape.

[0065] As shown in Figure 10B, the microfilter 1012 has a plurality of holes 1022, each having a width of 1042. The microfilter 1012 also has a thickness 1032 that is substantially perpendicular to the width 1042 of the holes 1022. The thickness 1032 of the microfilter 1012 is greater than the thickness of the microfilter 1010. In the embodiment shown in Figure 10B, the holes 1022 are uniform in size, and each is substantially perpendicular to the first surface 1050 and the second surface 1052 of the microfilter 1012. In the embodiment shown in Figure 10C, the microfilter 1014 has holes 1024 having a first width 1044 and holes 1026 having a second width 1046 that is smaller than the first width 1044. The microfilter 1014 also has a thickness 1034. In the embodiment shown in Figure 10D, the microfilter 1016 has holes 1028 with a non-uniform cross-sectional shape. Each hole 1028 has a first opening on the first surface 1054 of the microfilter 1016 and a second opening on the second surface 1056 of the microfilter 1016. As shown in Figure 10D, the width 1048 of the hole 1028 on the first surface 1054 is greater than the width 1049 of the hole 1028 on the second surface 1056. The microfilter 1016 also has a thickness of 1036. In the embodiment shown in Figure 10E, the microfilter 1018 has holes 1029 with a non-uniform cross-sectional shape. Each hole 1029 has a first opening 1045 on the first surface 1053 of the microfilter 1018 and a second opening 1047 on the second surface 1057 of the microfilter 1018. As shown in Figure 10E, the width 1045 of the hole 1029 on the first surface 1053 is smaller than the width 1047 of the hole 1029 on the second surface 1057. Microfilter 1018 has a thickness of 1038.

[0066] Figures 12A to 12K are cross-sectional views showing multiple steps in the manufacturing process of a multilayer microfilter 1270 according to an embodiment of the present invention. Figure 12L is a plan view of the multilayer microfilter 1270 according to an embodiment of the present invention. Figure 11 is a flowchart of the manufacturing process 1100 of a multilayer microfilter 1420 according to an embodiment of the present invention. Figures 14A and 14B are cross-sectional views of the multilayer microfilter 1420 according to an embodiment of the present invention. Figure 14C is a plan view of the multilayer microfilter 1420 shown in Figures 14A and 14B. Hereinafter, the exemplary process of Figure 11 will be described with reference to Figures 12A to 12F and Figures 14A to 14C. Figures 13A and 13B are plan views showing multiple steps in the process shown in Figure 11.

[0067] In block 1120 of Figure 11, a first microfilter 120 is formed on the substrate 180 from a layer of photodestructible epoxy film. In embodiments shown in Figures 12A to 12L, the first microfilter 120 may be formed on the substrate 180 by a process similar to process 200 in Figure 2A described above, omitting the removal of the microfilter 120 from the substrate 180 in block 280. In one embodiment, the microfilter 120 is composed of a polymer layer containing multiple apertures. In one embodiment of process 1100, instead of a mask 199 having a pattern 198 configured to form multiple holes in the dried film 100, a mask having a pattern configured to form multiple elongated trenches in the dried film 100 may be used. In such embodiments, the mask may have a pattern containing elongated metal strips, so that when the dried film 100 is exposed through the mask, corresponding elongated trenches are formed in the dried film 100.

[0068] Figure 13A is a plan view of a microfilter 120 formed in block 1120 according to an embodiment of the present invention. As shown in Figure 13A, the microfilter 120 includes a plurality of elongated trenches 1222 and is positioned on a substrate 180 that is exposed through the trenches 1222. Figure 12A is a cross-sectional view of the microfilter 120 cut along line 12A in Figure 13A, and Figure 12B is a cross-sectional view of the microfilter 120 cut along line 12B in Figure 13A. As shown, line 12B is perpendicular to line 12A.

[0069] In block 1140 of Figure 11, as shown in Figure 12C, a layer of epoxy-based photodestructible dry film 1210 (sometimes referred to herein as "dry film 1210") is placed over the microfilter 120. In one embodiment, the dry film 1210 can bridge structures formed on the surface beneath it. In such an embodiment, the dry film 1210 hardly penetrates the trench 1222 when placed over the microfilter 120. As will be described later, in block 1160, a second microfilter 1230 is formed from the layer of epoxy-based photodestructible dry film 1210. In one embodiment, the microfilter 1230 is composed of a polymer layer containing multiple apertures. As shown in Figure 12D, and as described above in relation to block 240 in Figure 2A, the dried film 1210 is exposed by energy through the mask 1290 to form an exposed dried film 1212 having a polymerized pattern 1218 and unpolymerized portions 1216. In the embodiments shown in Figures 12A to 12L, the dried film 1210 is a negative resist. In other embodiments, the dried film 1210 may be a positive resist, and different masks configured for use with a positive resist may be used. In the embodiments shown in Figures 12A to 12L, the dried film 1210 is exposed by energy in the form of ultraviolet (UV) light through an optical mask 1290 having a mask pattern 1293 including a mask portion 1295 transparent to UV light and a plurality of elongated strips opaque to UV light. In another embodiment, the dried film 1210 may be exposed to X-rays through an X-ray mask instead of being exposed to UV light through an optical mask 1290.

[0070] In the embodiments shown in Figures 12A to 12L, as described above in relation to block 260 in Figures 2A and 2B, in one modified example of block 1160, a polymer microfilter 1230 having a plurality of trenches 1232 is formed from the exposed dried film 1212. Here, the multiple trenches 1232 extend through the polymer microfilter 1230. Figure 13B is a plan view of the first and second microfilters 120 and 1230 according to an embodiment of the present invention. As shown in Figure 13B, the microfilter 1230 includes a plurality of elongated trenches 1232 and is positioned on the first microfilter 120 which is exposed through the trenches 1232. As shown in Figure 13B, the trenches 1232 of the microfilter 1230 are formed substantially perpendicular to the trenches 1222 of the microfilter 120. Figure 12E is a cross-sectional view of the microfilters 120 and 1230 cut along line 12E in Figure 13B, and Figure 12F is a cross-sectional view of the microfilters 120 and 1230 cut along line 12F in Figure 13B. As shown, line 12F is perpendicular to line 12E. The thickness of each layer may vary.

[0071] In one embodiment, after forming the second microfilter 1230, the substrate 180 may be removed from the microfilter 120 as described above in relation to block 280 in Figure 2A, to form the multilayer microfilter 1420 shown in Figures 14A to 14C. In the embodiments shown in Figures 14A to 14C, the multilayer microfilter 1420 includes the second microfilter 1230 formed on the first microfilter 120. As shown in Figure 14C, the multilayer microfilter 1420 includes an aperture 1240. This aperture 1240 extends to the portion of the multilayer microfilter 1420 where the trenches 1222 and 1232 intersect. In one embodiment, the microfilter 1240 is composed of a polymer layer containing a plurality of apertures. Figure 14A is a cross-sectional view of the microfilter 1420 cut along line 14A in Figure 14C, and Figure 14B is a cross-sectional view of the microfilter 1420 cut along line 14B in Figure 14C. As shown in the diagram, line 14B is perpendicular to line 14A. The thickness of each layer may be different.

[0072] In one embodiment, instead of forming the microfilter 1420, a microfilter 1270 having a nonlinear passage 1280 may be formed, as shown in Figures 12G to 12L. In such an embodiment, the multilayer microfilter 1270 shown in Figure 12L is formed by forming a third microfilter 1240 on the first and second microfilters 120 and 1230 and removing the substrate 180. In another embodiment, as shown in Figure 12F, the second microfilter 1230 is formed on the first microfilter 120 and the substrate 180, and then, as shown in Figure 12G, a layer of photodeterminable epoxy-based dry film 1215 is applied to the second microfilter 1230. Subsequently, the third microfilter 1240 is formed from the dry film 1215 as described above in relation to the formation of the second microfilter 1230 and the processes of blocks 240 and 260 in Figures 2A and 2B. As shown in Figure 12H, the third microfilter 1240 includes a plurality of elongated trenches 1242 that are substantially perpendicular to trench 1232 and substantially parallel to trench 1222. In one embodiment, the trenches 1242 are formed offset from the position of trench 1222, so that the trenches 1242 are not located directly above trench 1222, as shown in Figure 12H.

[0073] In one embodiment, after forming the third microfilter 1240, the substrate 180 may be removed from the microfilter 120 as described above in relation to block 280 in Figure 2A to form the multilayer microfilter 1270. Figure 12L is a plan view of the multilayer microfilter 1270. Figure 12J is a cross-sectional view of the multilayer microfilter 1270 cut along line 12J in Figure 12L, and Figure 12K is a cross-sectional view of the multilayer microfilter 1270 cut along line 12K in Figure 12L. As shown, line 12K is perpendicular to line 12J.

[0074] As shown in Figure 12L, the multilayer microfilter 1270 includes a nonlinear passage 1280 extending through each of the microfilters 1240, 1230, and 120. This nonlinear passage 1280 extends from a first surface 1272 to a second surface 1274 (see Figure 12J) of the multilayer microfilter 1270. In one embodiment, each nonlinear passage 1280 is defined by a first aperture 1282 at the intersection of trenches 1242 and 1232, a second aperture 1284 at the intersection of trenches 1232 and 1222, and a portion of trench 1232 connecting the first and second apertures. In embodiments in which the multilayer microfilter 1270 has one or more nonlinear passages 1280, the filtration path is longer than when the multilayer microfilter 1270 contains only linear apertures. For clarity, Figure 12L shows only the selected trenches 1232 and 1222 and the selected nonlinear passages. In some embodiments, each nonlinear aperture 1280 is interconnected with many other nonlinear passages 1280 via the trench 1232.

[0075] In one embodiment of the multilayer microfilter 1270, the thicknesses of the microfilters 120, 1230, and 1240 may be the same or different. Furthermore, the trenches of the microfilters do not all have the same size and / or shape, and the trenches of different microfilters in the multilayer microfilter do not all have the same size and / or shape. In some embodiments, the elongated trench 1242 may have a width of 5 to 7 microns and a length greater than 7 microns, where the length and width are perpendicular to the thickness of the microfilter. Alternatively, or further, the trenches of the microfilter thickness may be nonlinear, and the trenches of adjacent microfilters may be arranged at angles other than 90 degrees to each other. Alternatively, or further, one or more of the microfilters 120, 1230, and 1240 may contain holes similar to any of the holes shown in Figures 9A to 9D instead of trenches, and in some embodiments, the multilayer microfilter 1270 may contain four or more microfilters formed on top of each other. In one embodiment, each of the microfilters 120, 1230, and 1240 may be formed from the same type of photodeterminable epoxy-based dry film.

[0076] Figure 15 is a cross-sectional view of a microfiltration structure including a microfilter and a support structure according to an embodiment of the present invention. In the embodiment shown in Figure 15, the microfiltration structure 1510 has a microfilter 1520 having pores 1522, and the microfilter 1520 is disposed on a support structure 1530 configured to provide structural strength to the microfilter. In some embodiments, the support structure 1530 may be integrated with the microfilter 1520. In some embodiments, the support structure 1530 is a grid-like support structure. In some embodiments, the microfiltration structure 1510 may be formed by a process similar to the process described above in relation to Figures 12A to 12F and Figures 14A to 14C. In such embodiments, each of the microfilter 1520 and the support structure 1530 is formed from a layer of photodeterminable dry film of an epoxy system and its shape is determined using an appropriate mask. In such embodiments, the microfilter 1520 is formed on the support structure 1530, or the support structure 1530 is formed on the microfilter 1520.

[0077] Microfilters can be a combination of pores and other structural elements above or below the pore-forming layer to form many filtration devices. Several examples of two- or three-layer structures, with or without pores, are shown in Figures 16A–16J. These devices have applications for separating cells from bodily fluids and for biological analysis.

[0078] Figure 16A shows a typical embodiment of a two-layer microfilter, a side view of a microfilter 2561 including posts 2575 on a microfilter base 2572 along with holes 2573. Figure 16B shows a plan view of posts 2575 on a microfilter base 2572 along with holes 2573. The holes can be circular, square, rectangular, etc. The posts can also be of various shapes, such as circular, square, rectangular, etc. The arrangement and density of the posts may be regular, spatially varied, or random, as long as the distance between posts is no more than 30 μm and the posts do not cover the holes. The holes can have various shapes, sizes, and distributions. The posts can also have various shapes, sizes, and distributions.

[0079] Figure 16C shows a plan view of microfilter 2563, which is another exemplary embodiment of a two-layer microfilter, in which the pores are formed in a rectangular shape 2573 but are not distributed anywhere on the base support 2572, nor are the posts 2575 distributed anywhere. The pores can have various shapes, sizes, and distributions. The posts can also have various shapes, sizes, and distributions.

[0080] Figure 16I shows an exemplary embodiment of a two-layer apparatus 2580, with a side view of the non-perforated bottom layer 2581. The top layer 2582 forms a well 2583. Figure 16J shows a top view. This well structure can be manufactured using the same procedure described in Figure 1A-1D, following the procedure described in Figures 12A-12F, except that the mask shown in Figure 1B is not required.

[0081] Pore ​​dimensions vary depending on the specific application requirements. For example, pores with a diameter of 7-8 microns are generally preferred for circulating tumor cells from human blood, but smaller pores are preferred for mouse studies because circulating tumor cells derived from mice are smaller than those from humans. For rectangular pores, a width of 5-7 μm is preferred for collecting circulating tumor cells from human blood, and the length is not important as long as the filter does not deform during filtration.

[0082] Figures 16D-16F show another exemplary embodiment of a two-layer microfilter having different patterns in each layer of material, according to an exemplary embodiment. Figure 16D shows a first side view 2501 of a two-layer microfilter having an upper layer 2551 and a bottom layer 2552. The upper layer is a strip having slot openings 2555. Figure 16E is a second side view 2502 of the two-layer microfilter rotated 90 degrees. The bottom layer 2552 has empty slots 2553. Figure 16F shows a plan view of the microfilter 2500. The cross strip effectively forms rectangular holes 2554.

[0083] In an exemplary embodiment, the dimensions of the upper channel 2555 between the two upper strips 2551 are 5–7 μm for the separation of tumor cells. A small width of 10–20 μm for 2551 allows for high filtration, but a wider range for 2551 would be functional. The width of 2552 can be varied, but for high filtration, the width can be 5–20 μm. The gap between the two strips of 2552 that constitute the pore 2553 can also be varied. For high filtration, the gap can be 10–60 μm. A thickness of about 10 μm for 2551 may be preferred. In an exemplary embodiment, the thickness of the bottom layer 2552 can advantageously be approximately the same as the gap between the two strips of 2552.

[0084] Figures 16G-16H are side and top views of an exemplary apparatus 2540 consisting of a microfilter 2541 having holes 2542 at the bottom of wells 2544. The structure shown in Figure 16H is repeated throughout the entire microfilter area. The application of apparatus 2540 is to filter cells and subsequently culture the cells in wells 2544. Again, the holes can have various shapes and sizes. The size, shape, and depth of wells 2544 can be changed to suit different applications. The density of wells 2583 can also be changed.

[0085] Exemplary applications of devices 2500, 2540, 2561, and 2563 include the isolation of rare cells when the cell isolation mechanism is size-based.

[0086] Figures 16I-16J are side and top views of an exemplary apparatus 2580 consisting of a well 2583 formed by a solid bottom 2581 2582. These can be used for various biological analyses. The size, shape, and depth of the well 2583 can be varied. The density of the well 2583 can also be varied.

[0087] The various materials for forming the apparatus described in Figures 16A-16J above may include epoxy-based photo-deficient dry films and other types of photo-deficient dry films. Methods for fabricating these structures using photo-deficient dry films have been described above and, for example, in PCT / U.S. Patent Application 11 / 20966. <Coating of microfilters>

[0088] In certain embodiments, surface functionalization of polymer microfilters can provide a microfilter surface having surface properties desired for a particular application of the microfilter. Some materials can be directly placed on the microfilter surface. In other cases, the microfilter surface needs to be treated. In one embodiment, the surface of a polymer microfilter can be functionalized by plasma treatment of the microfilter surface to activate the surface so that chemical compounds and / or organic materials can adhere to it. In some embodiments, another surface modification technique is to coat the microfilter with a thin layer of metallic material.

[0089] Figures 17 and 18 are cross-sectional views of coated microfilters according to embodiments of the present invention. In the embodiment shown in Figure 17, the microfilter 1655 includes a coating 1600 on the surface of one layer of the microfilter 1655. The coating 1600 can also be placed on a multilayer microfilter. The coating 1600 can also be placed on a microfilter manufactured by other methods and from other materials. In Figure 18, as described above in relation to Figures 16A-16J, the multilayer device is formed from an epoxy-based photodeterminable dry film. In the embodiment shown in Figure 18, the microfilter 1672 includes a coating 1600 on the surface of the superstructure and a flat portion of the microfilter 1672. The coating 1600 can also be placed on a microfilter structure manufactured by other methods and from other materials. The coating 1600 can also be placed on the surface of the structure shown in Figures 16I-16J, even if it is not perforated.

[0090] In certain embodiments, the coating 1600 can be formed from a metallic substance, a nanoparticle colloidal substance, a chemical substance, or an organic substance. In such embodiments, these surface coatings can be used to attach analyte recognition elements, DNA, aptamers, surface blocking reagents, etc. In other embodiments, the coating 1600 may include analyte recognition elements, DNA, aptamers, surface blocking reagents, etc. In certain embodiments, the surface coating can be used to attach macromolecules such as peptides, nucleic acids, carbohydrates, and lipids. Examples of polypeptides that can be used as analyte recognition elements include, for example, antibodies, antigen targets for antibody analytes, receptors (including cell receptors), binding proteins, ligands, or other affinity reagents for target analytes. Examples of nucleic acids that can be used as analyte recognition elements include, for example, RNA, DNA, or cDNA of any length that allows sufficient binding specificity. In such embodiments, both polynucleotides and oligonucleotides can be used as analyte recognition elements. In other embodiments, gangliosides, aptamers, ribozymes, enzymes, antibiotics, or other chemical compounds can be used as analyte recognition elements. In certain embodiments, the sample recognition coating or element includes biological particles such as cells, cell fragments, viruses, bacteriophages, or tissues. In some embodiments, the sample recognition coating or element may include BSA, fetal bovine serum (FBS), selectins including P-selectin, E-selectin, and L-selectin, nanoparticles, nanotubes, halloysite, dendrimers, chemical linkers, or other chemical moieties that can be attached to a microfilter and exhibit selective binding activity toward the target sample.

[0091] In some embodiments, the coating 1600 can be formed from a metallic substance such as gold or nickel. In certain embodiments, this includes gold coated on chromium. In some embodiments, it may be preferable to form the coating 1600 from gold as a certain chemical compound and organic material that readily adheres to gold. In other embodiments, the coating 1600 can be formed from carbon nanotubes. In the embodiments shown in Figures 17-18, the coating 1600 is placed on one surface of the microfilter. In other embodiments, one or more surfaces of the microfilter can be coated with the coating 1600. In some embodiments, the microfilter can be completely coated with the coating 1600. The coating 1600 can be placed on one or more surfaces of any microfilter described in embodiments of the present invention, including multilayer microfilters. In certain embodiments, the coating 1600 can be placed on one or more surfaces of a multilayer microfilter 1620.

[0092] In some embodiments, examples of chemical compounds and organic materials that may be useful for analysis when deposited on the surface of a microfilter include self-assembled monolayers with a range of functionalities, including the groups of amines, carboxyls, hydroxyls, epoxys, aldehydes, and polyethylene glycol (PEG). These compounds and materials may be deposited on the surface of the microfilter using silane chemistry by solution immersion or vapor deposition. In certain embodiments, for example, grafting PEG-triethoxysilane onto an oxidized polymer renders a hydrophilic surface in a controlled manner. In other embodiments, the surface of a polymer microfilter can be functionalized with avidin, biotin, protein A, protein G, antibodies, etc.

[0093] In certain embodiments, coating the surface of a microfilter with a metallic substance can offer other advantages in addition to facilitating the adhesion of chemical compounds and / or organic materials. In some embodiments, for example, a layer of metallic substance of appropriate thickness can block the transmission of light through the microfilter. In certain embodiments, a thickness sufficient to block light transmission is approximately 40 nm. In other embodiments, this thickness may vary depending on the material used. Furthermore, metallic substances are generally conductive. In some embodiments, when the metallic substance is conductive, the coating can reduce or eliminate the charge on the surface of the microfilter. In alternative embodiments, the microfilter may be coated with a thin layer of PARYLENE. In other embodiments, the microfilter can be coated with a thin layer of fluorinated ethylene propylene (FEP), polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), or other similar materials. In such embodiments, coating the microfilter with one of these materials can reduce nonspecific bonding; however, if the microfilter is analyzed by microscopic imaging, the fluorescence properties of these materials can be detrimental to them.

[0094] In certain embodiments, single-layer or multi-layer microfiltration devices can be coated with antibodies against surface markers on CTCs to further improve the recovery of live CTCs. In such embodiments, the capture efficiency of live CTCs can be improved. In some embodiments, useful surface markers may include, but are not limited to, antibodies against EpCAM, HER2, EGFR, KRAS, vimentin, and MUC-1, and may include P-selectins, E-selectins, other selectins, ligands, aptamers, etc. In embodiments where the microfilter is coated with cell surface recognition elements, microfiltration can simultaneously capture CTCs via size exclusion and surface markers. <Filter holder and filtration device>

[0095] If a microfilter can be installed in a precision filtration system, the filtration process of large, rare cells from body fluids concentrated with reduced amounts of contaminants on or off the microfilter can be performed with minimal human intervention. This invention describes a method of using a microfilter with a filter holder.

[0096] As shown in Figure 19, microfilters made from epoxy-based photodestructible dry films can be used in a variety of methods for analyzing rare cells such as CTCs from bodily fluids. Rare cells can be collected on epoxy-based photodestructible dry film microfilters or other precision microfilters. Following the collection of rare cells, they can be analyzed using the following methods. • Enzyme analysis in rare cells such as CTCs, which can be tested to evaluate viability and enzyme activity; • Histopathological staining to observe cell morphology; Immunofluorescence staining can be used to examine the expression of biomarkers to identify cancer subtypes, or to determine biomarker mutations or cell types; this information can be used to determine and monitor cancer treatment; immunofluorescence staining can be used to enumerate CTCs based on DAPI (positive), cytokeratin (CKs) (positive), e.g., CK8, 18, 19, etc., and CD45 (negative) cells, and to count the number per milliliter of blood to monitor cancer treatment and recurrence; • Fluorescence in-situ hybridization (FISH) to identify cancer subtypes, to determine and monitor treatment, to identify gene amplification, replication of biomarker genes, and the number of gene translocations; mRNA FISH can also be used to determine if markers are overexpressed. • Nucleic acid analysis for mRNA, microRNA, and genomic DNA biomarkers, gene mutations to identify cancer subtypes, determine and monitor treatment; • Gene sequencing to identify gene mutations, amplifications, and translocations in order to determine and monitor treatment, identify cancer subtypes; • Culturing of rare cells such as CTCs to increase the number of cells. These cells can then be used to perform the analyses described in the bullet points above. In addition, surviving cells can be used to determine the effect of drugs on cancer cells in order to determine treatment. • Culture of rare cells in microfilters coated with a sample / marker recognition element for samples / markers that can be secreted by CTCs. Analysis similar to that of EPISPOT can be performed using microfilters.

[0097] Some of these analytical methods can be performed in combination or sequentially on rare cells collected on an epoxy-based photodestructible dry film. For example, rare cells collected on an epoxy-based photodestructible dry film can be performed sequentially to obtain different information from the same cells. Some examples include (1) performing enumeration for cancer surface biomarkers following FISH analysis and final histopathological staining, (2) counting the number of rare cells following histopathological staining and simultaneously performing immunofluorescence staining to determine biomarker overexpression, mutated biomarkers, cell type, and / or other information, and (3) performing FISH to identify markers following histopathological staining.

[0098] According to exemplary embodiments of the present invention, a filtration apparatus for holding a filter and performing filtration is designed to have the following features: A filter holder for holding filters flat without causing twisting. This filter holder has a large opening in the inlet unit. This allows for easy access of reagents to the microfilter surface and visual access to the microfilter once cells have been collected, and enables visualization of the filter from above. The filter holder enables at least some kind of performance in the analysis performed by the filter holder. The filter holder has an input sample holder on top, and a stopper for filter flasks connected to a vacuum pump or vacuum container holder, as well as various syringe mounting options below.

[0099] According to one embodiment of the present invention, the filter holder can be for the following: (i) the ability to filter a liquid sample through a microfilter, (ii) the ability to perform analysis with the microfilter in the holder, (iii) easy access to the microfilter in the filter holder, (iv) easy installation of the microfilter into the filter holder, (v) easy removal of the microfilter from the filter holder, (vi) a filter holder material capable of handling the permissible temperature required for the analysis, and (vii) a filter holder material capable of handling the permissible chemicals used in the analysis.

[0100] Figures 20A-20C show three components forming a filter holder 3000 according to one embodiment of the present invention. It includes an inlet unit 3010, a nut 3020, and an outlet unit 3030 (Figure 20C in a top view and Figure 20D in a bottom view). In an exemplary embodiment, a syringe or a vacuuminer holder with a Luer lock can be attached to the outlet unit of the filter holder. In an exemplary embodiment, the outlet unit may include, for example, a notch on the top ring to allow access to the microfilter in order to enable easy removal of the microfilter from the filter holder.

[0101] Figures 21A-21E show an assembly of a filter holder 3100 according to one embodiment of the present invention, with a gasket 3110 positioned inside the outlet unit as shown in Figure 21A. A circular microfilter 3120 having an appropriate diameter for the filter holder is positioned on top of the gasket as shown in Figure 21B. A second gasket 3130 is positioned on top of the microfilter 3120 as shown in Figure 21C. The inlet unit 3010 is positioned inside the outlet unit 3030 with the two parts properly aligned as shown in Figure 21D. Finally, a nut 3020 is installed to tighten the filter holder system around the microfilter in the holder to prevent leakage of the liquid sample. One gasket may suffice, but multiple gaskets may be used as needed, for example, to prevent leakage. The gaskets may be of different materials or designs. In the exemplary filter holder design, the microfilter positioned in the filter holder system will remain flat and will not experience any torsional forces, although it will not experience any compression to form a seal.

[0102] There are many potential variations of the filter holder that remain within the scope of the exemplary embodiments of the present invention, including, but are not limited to, the following: Filter holders can have different sizes to accommodate microfilters of different sizes. Common microfilter sizes are 0.5 inches (13 mm) and 1 inch in diameter, but are not limited to these dimensions. The filter outlet unit may have one or more openings. The openings allow for easy removal of the microfilter from the filter holder and prevent the microfilter from twisting by securing the inserted inlet unit. The shape and dimensions of the different parts of the filter holder can be changed. One of the features of the filter holder concept according to one embodiment of the present invention is that it pushes down the inlet unit to prevent leakage. In an exemplary embodiment, a nut is used to keep the inlet unit pressed down. The entrance unit can be kept pressed down by changing the nut on the snap-on part. The outlet 3030 may have a female connector or a male connector. The filter holder can be designed to accommodate one gasket below the microfilter, or two gaskets, one above the microfilter and one below the microfilter. • The filter support structure can be added inside the output unit of the filter holder. Since the material is strong enough, the support structure may not be necessary for microfilters of photodestructible dry films using epoxy-based materials. The entrance unit 3010 can be combined with the entrance adapter 3050 shown in Figure 23 to form a single unit.

[0103] The exemplary embodiments of the filter holder described in this application are applicable to most filters and microfilters. Certain exemplary embodiments are particularly suitable for very thin, strong microfilters, such as epoxy-based photodestructible dry film microfilters.

[0104] Figures 22A and 22B show examples of sample input containers for holding liquid samples and wash buffer. Figure 22A shows an inlet container 3200 with a syringe-like shape having a sample inlet opening 3210 and a connection to a filter holder 3220. Figure 22B shows an inlet container 3300 with a wider opening 3310 and a connection to a filter holder 3320 that allows easy access to a microfilter. Figures 22A and 22B show examples of integrated sample input containers.

[0105] Figure 23 shows an example of a sample input container 3420 that can be constructed from a commercially available syringe 3410 without an inlet adapter 3050 having a plunger and a male Luer lock. The inlet adapter can have various designs, as long as it does not leak and can be easily removed from the filter holder. With the same bottom shape as the sample input containers 3200 and 3300, the inlet adapter 3050 should have a shape to facilitate a tight fit into the inlet unit 3010 on top of the filter holder 3100 assembled in Figure 21E. Alternatively, or in combination, an O-ring can be provided to facilitate a tight fit into the inlet unit 3010.

[0106] The filtration system 3500 can take the configuration shown in Figure 24. In this example, the input container 3420, configured as shown in Figure 23, is connected to the top of the filter holder 3100 assembled from Figure 21E, and the bottom of the assembled filter holder 3100 shown in Figure 21E is connected to the waste syringe 3510 having a plunger 3520.

[0107] Figure 25 shows the filtration system with the filter holder 3100 and the waste syringe 3510. The opening 3140 allows for the performance of many analytical processes directly on the microfilter.

[0108] Filtration can be performed by manually pulling the plunger, but manual operation may not provide a consistent speed. The filtration system 3600 using the syringe pump 3610 in Figure 26 can provide a more consistent speed for pulling the plunger 3520 by the pusher block 3620. The syringe pump can have the function of injecting only, or both injecting and recovering.

[0109] Figure 27A shows another exemplary method for drawing blood through a microfilter by connecting a vacuuminer holder 3710 to the outlet of a filter holder 3730. As shown in Figure 27B, when a vacuuminer 3740 is inserted into the vacuuminer holder 3710, the vacuum in the vacuuminer will draw the liquid sample into the vacuuminer, and the microfilter will collect rare cells.

[0110] Figure 28A shows a filtration device 3800 that can perform filtration at the time of blood collection. The filtration device combines a filter holder 3830 having an inlet adapter 3820 with a vacuum container holder 3810. A needle 3850 with a female Luer lock can be attached to the inlet adapter 3820. Figure 28B shows a vacuum container 3840 inserted into the vacuum container holder 3810. <Performing sample collection and analysis using a filtration system>

[0111] A logistics process for collecting a sample and sending it to a clinical laboratory for rare cell analysis, according to one embodiment of the present invention, will be described below with reference to Figures 29A and 29C.

[0112] Figure 29A shows an example of a method for obtaining and sending a sample to a clinical laboratory for testing. The test includes: (i) collecting body fluids, (ii) sending the sample to the laboratory, (iii) filtering rare cells from the body fluids, and (iv) performing the analysis.

[0113] Because cells may degrade in quality, blood may clot, and the time required for shipment may be 24-48 hours, Figure 29B describes an alternative method for obtaining samples and testing them in a clinical laboratory. It includes: (i) collecting body fluids, (ii) filtering rare cells from the body fluids in the collection room, (iii) sending the filter holder with a microfilter, or the filter holder alone with the captured rare cells, to the laboratory, and (iv) performing the analysis in the laboratory.

[0114] For blood samples, it is also possible to filter rare cells from the blood at the time of collection using the filtration device 3800 shown in Figure 29C. After the washing process, the filter holder together with the microfilter, or just the filter holder together with the captured rare cells, can be sent to the laboratory. Analysis will then be performed in the laboratory. <Applications of microfilters>

[0115] In the embodiments of the present invention described above, the microfilter may be formed from an epoxy-based photodestructible dry film having a thickness of 1 to 500 μm. In some embodiments, such a microfilter may be formed using UV light (i.e., UV lithography) to expose the dry film. In some embodiments, X-rays (i.e., X-ray lithography) may be preferred for exposure of relatively thick dry films, simultaneous exposure of multiple laminated dry films, or exposure of resists requiring relatively high irradiation doses. In some embodiments, a relatively thick microfilter may have higher structural strength than a thinner microfilter, but it may be necessary to use higher pressure during filtration.

[0116] As described above, in some embodiments, an epoxy-based photodestructible dry film is a preferred material for forming a microfilter according to the embodiments of the present invention. In some embodiments, properties of an epoxy-based photodestructible dry film that make it a suitable material for forming a microfilter for medical diagnostic applications include being photodestructible with UV light, being clear, having a high tensile strength of 75 MPa, being bondable, being able to be directly coated onto a substrate, and not exhibiting autofluorescence at visible light wavelengths. Furthermore, while the process described above according to the embodiments of the present invention can be used to form microfilters, it can also be used to manufacture other types of independent, patterned polymer films.

[0117] Microfilters formed according to embodiments of the present invention can be used for a wide range of applications. In some embodiments, examples of applications for such microfilters include medical applications, water filtration, beer and wine filtration, and applications for pathogen detection.

[0118] Figure 30A is a flowchart of a filtration process 1700 using a microfilter according to an embodiment of the present invention. In block 1720 of Figure 30A, a liquid may be flowed through a microfilter having multiple apertures formed from layers of photodestructible dry epoxy film according to any of the embodiments described above. In some embodiments, the liquid may be forced through the microfilter. In other embodiments, the liquid may be drawn in through the microfilter. Drawing in can be done by a syringe or by vacuum. In some embodiments, the liquid may be flowed so as to pass back and forth (back and forth) through the microfilter one or more times. In some embodiments, the fine particles retained on the microfilter can be backwashed with a suitable liquid.

[0119] In one embodiment, the process shown in Figure 30A may be used to perform an analysis using a microfilter. In another embodiment, this process may be used to filter cells such as CTCs from a solution containing a patient's bodily fluids. This can be achieved using a vacuum container or by placing the filter on a support above a vacuum pump, as shown in Figures 28A-28B.

[0120] Figure 30B is a flowchart illustrating a filtration process 1701 using a microfilter according to an embodiment of the present invention. In block 1830 of Figure 30B, a microfilter formed from a layer of photodeterminable dry film of an epoxy system according to any of the embodiments described above is positioned in a filter holder. In one embodiment, the filter holder includes an inlet and an outlet and securely holds the microfilter around its outer circumference. In some embodiments, a liquid may be introduced into the filter holder through the outlet. In block 1850, the liquid flows through the microfilter. In one embodiment, the liquid is a body fluid or a solution containing a body fluid. In one embodiment, the liquid is drawn through the microfilter by applying negative pressure to the outlet of the filter holder so that all or almost all of the liquid is drawn through the pores of the microfilter. In other embodiments, the liquid may be forced through the microfilter. In block 1870, the microfilter is removed from the filter holder. For microscopic imaging, the microfilter may be placed on a glass slide.

[0121] Figure 30C is a flowchart 1900 showing an example of performing several types of analysis on the filter holder 1910. After the analysis, the filter can be removed from the filter holder 1920 for analysis.

[0122] Enzyme activity analysis, histopathological staining (colorimetric staining), and immunofluorescence staining for applications such as biomarker expression, EPISPOT, and enumeration determination can be performed following the steps shown in Figure 30B, followed by the flowchart in Figure 30C. These analyses can be performed in the filter holder as described. These analyses can also be performed on a glass slide or plate after removing the microfilter from the filter holder, following the flowchart shown in Figure 30A or 30B.

[0123] Nucleic acid analysis, sequencing, fluorescence in-situ hybridization (FISH), mRNA in-situ hybridization, and culture require the removal of the microfilter from the filter holder along with the captured rare cells, and the analysis is performed using the appropriate method for each analysis, as shown in the flowchart in Figure 30A or 30B.

[0124] In the illustrative flowchart of Figure 30B, the protocol for separating cells using a syringe pump is as follows: 1. Assemble the microfilter into the filter holder as shown in Figure 21-21E. 2. Mount the waste syringe 3510 onto the syringe pump 3610. 3. Mount the assembled filter holder 3100 onto the waste syringe 3510. 4. Attach the inlet container 3420 (Figure 22A, 22B, or 23) on top of the filter holder 3100 to assemble the complete unit as shown in Figure 26. 5. Place the liquid sample in the inlet container 3420. 6. Use negative pressure to draw the liquid sample into the waste syringe via the microfilter. 7. To perform the washing process, place the washing buffer into the inlet container 3420 and draw the washing buffer into the outlet syringe via the microfilter. Perform several washes. 8. Remove the inlet container 3420, and the filtration system will look like Figure 25. 9. Open the filter holder to retrieve the microfilter.

[0125] Syringe pumps can be operated manually or automated.

[0126] Filtration can be performed manually. To perform the analysis manually, skip step 2. At the start, the completed assembly system will look like Figure 24.

[0127] The filter holder 3100 can be connected to a vacuum pump equipped with an inlet container 3420 and placed on the stopper of the filter flask. The liquid sample and washing buffer can be placed in the inlet container 3420. The vacuum pump is turned on to draw the liquid through the filter. If the inlet container is not used, the liquid sample and washing buffer may be pipetted into the reaction well 3010 while the vacuum is on.

[0128] Filtration can be performed using the Vacutainer System 3700, as shown in Figure 27A. The procedure is as follows: 1. Assemble the microfilter inside the filter holder as shown in Figures 21A-21E. 2. As shown in Figure 27A, attach the vacuuminer holder to the outlet of the filter holder 3730. 3. Attach the input container 3720 (Figure 22A, 22B, or 23) on top of the filter holder 3730, and the completed assembly will look like Figure 27A. 4. Place the liquid sample in the input container 3720. 5. Insert the vacuum container 3740 into the vacuum container holder 3710, as shown in Figure 27B. 6. Perform the washing step to place the washing buffer into the input container 3720. 7. Install a fresh vacuum container 3740 into the vacuum container holder 3710 for cleaning. 8. For further processing of the cells, collect them on a microfilter or open the filter holder to retrieve the microfilter, as needed.

[0129] Filtration can be performed at the time of blood collection using the Vacutainer System 3800, as shown in Figure 28A. The procedure is as follows: 1. Assemble the microfilter into the filter holder as shown in Figures 21A-21E. 2. As shown in Figure 28A, attach the vacuum container holder to the outlet of the filter holder. 3. Attach the needle 3850 to the entrance adapter 3820 using a female Luer lock. 4. Insert the needle into the patient's vein. 5. Insert the vacuum container 3840 into the vacuum container holder 3810, as shown in Figure 28B. 6. After retraction, remove the Vacutainer 3840. 7. Remove the needle from the patient's vein. 8. Remove the needle from the filter holder and install the input container 3720 as shown in Figure 27. 9. Perform the washing process and place the washing buffer into the input container 3720. 10. As shown in Figure 27B, install a fresh vacuum container 3740 into the vacuum container holder 3710 for cleaning. Insert a syringe into the outlet of the filter holder as shown in Figure 23. 11. For further processing of the cells, collect the microfilters as needed, or open the filter holder to retrieve the microfilters.

[0130] Filters with attached cells can be used for various tests and analyses.

[0131] Figure 30C shows that numerous analytical steps can be performed in the filter holder after each step in the flowcharts in Figures 30A-30B. The details of the analytical steps may vary depending on the analysis. This is done using the configuration shown in Figure 25. The steps will vary depending on the analysis. A general procedure is described below. 1. Culturing: Place the reagents in reservoir 3140 on the filter and begin culturing. Because the epoxy-based photocurable dry film microfilter is hydrophobic, it is possible to culture the reagents on the microfilter in the filter holder. The reagents will not leak through the microfilter. 2. Washing: For a small amount of washing buffer after culture, the washing buffer can be placed directly into reservoir 3140 and aspirated by negative pressure. This can be repeated. If a larger amount of washing buffer is needed, the inlet container is placed back on top of the filter holder to form the system shown in Figure 24, and the washing buffer placed in the inlet container is aspirated to perform the washing process. 3. Whenever the outlet syringe becomes full, replace it with a new one. 4. Once the analysis is complete, disassemble the filter holder, remove the microfilter, and place it on a glass slide, plate reader, or other suitable device for image analysis.

[0132] Nucleic acid analysis and sequencing are suitable for analysis performed from lysed cells. The conceptual protocol follows the procedure for collecting cells on a microfilter. After removing the microfilter from the filter holder, place the microfilter containing the rare cells i6 in an Appendorf centrifuge tube with lysis buffer. The remaining procedure for nucleic acid analysis is the same as for general samples.

[0133] To culture rare cells, the conceptual protocol follows the step of harvesting cells on a microfilter. After removing the microfilter from the filter holder, the microfilter containing the rare cells is placed in the culture medium. The cells can also be backwashed from the filter into the culture medium. In some situations, the rare cells need to be removed from the microfilter and injected into animals such as mice. <Analysis of filtered cells>

[0134] In some embodiments, the particles subsequently held on the microfilter may be subjected to processing or analysis for the analysis of any cells or other materials, substances, etc., collected by the microfilter. The analysis can be performed in a filter holder device or a microfiltration tip. After the microfilter is removed from the filter holder, the analysis may also be performed outside the filter holder. Exemplary applications of this process according to embodiments of the present invention are described below.

[0135] In some embodiments, a microfilter formed from a layer of photodestructible dry epoxy film according to any of the embodiments described above may be used for medical diagnostics and / or prognostics. In some embodiments, the microfilter may be used to collect certain types of cells from body fluids based on cell size. In some embodiments, the microfilter may be used to isolate and detect rare cells from biological samples containing other types of cells. In some embodiments, the microfilter may be used to filter fluid samples, and the collected cells may be used in downstream processes such as cell identification, counting (counting the number of cells), characterization of collected cells, culture of collected cells, separation into individual cells or cell populations, or other uses of the cells. The target cells, whose concentration has been ultimately increased, can be subjected to various characterization studies and manipulations, including staining, immunofluorescence markers, cell counting, DNA analysis, mRNA analysis, microRNA analysis, fluorescence in-situ hybridization (FISH), immunohistochemistry, flow cytometry, immunocytochemistry, image analysis, enzyme analysis, gene expression profiling, sequencing, therapeutic efficacy testing, culture of concentrated cells, and therapeutic use of concentrated rare cells. Furthermore, deficient plasma proteins and leukocytes can be optionally recovered and subjected to other analyses such as inflammation studies and gene expression profiling.

[0136] In some embodiments, the microfilter may be held in a filter holder for medical diagnosis and / or predictive diagnosis. In some embodiments, the filter holder may include a built-in support for the microfilter. In some embodiments, the filter holder may have gaskets above and below the filter. In some embodiments, the microfilter may be used to collect circulating tumor cells (CTCs) in the blood. In such embodiments, a blood sample in the range of 1 to 10 ml is typically taken from the patient. The blood sample is then drawn through the microfilter by applying negative pressure, such as suction. In some embodiments, the blood is drawn into the microfilter through an outlet. In some embodiments, forcing blood through the filter may cause cell destruction unless done at very low pressure or slow speed.

[0137] In one embodiment, most cells with dimensions greater than the width of one or more pores in the microfilter are retained. Most leukocytes can deform and pass through pores narrower than their dimensions. In one embodiment, red blood cells are hardly retained on the microfilter. In some embodiments, the microfilter may contain pores with a diameter of 7-8 μm to increase the concentration of circulating tumor cells and embryonic cells, but the size and shape of the pores in the microfilter can also be modified depending on these applications.

[0138] In some embodiments, CTCs collected by a microfilter can be counted on the microfilter. In one experiment performed to determine the capture efficiency of the microfilter, a tumor cell line was used. The microfilter used to demonstrate the filtration efficiency was a microfilter with pores 7-8 microns in diameter arranged within a 9 mm diameter area at 20 micron intervals. This microfilter was placed in a filter holder. A colored MCF-7 cell line was added to 7.5 ml of whole blood. To capture live CTCs, the blood was diluted 1:1 with a buffer solution. One exemplary buffer solution is phosphate-buffered saline (PBS). Another representative buffer solution is a loose fixation buffer that makes the CTCs slightly stiffer. The liquid sample was drawn into the microfilter at approximately 10 ml / min using negative pressure. The filter was then washed twice in the buffer solution. The microfilter was removed from the holder and placed on a microscope slide for counting. The recovery rate of live MCF-7 cells was 85% ± 3%. Lightly fixing the blood with paraformaldehyde increases the capture efficiency of MCT-7 cells to 98% ± 2%.

[0139] In some embodiments, collected CTCs can be subjected to various analyses and manipulations, including immunofluorescence, genetic characterization and molecular phenotyping, fluorescence in-situ hybridization (FISH), mRNA FISH, in-situ hybridization (ISH), mRNA FISH, immunohistochemistry (IHC), flow cytometry, immunocytochemistry, colorimetric staining, histopathological staining (e.g., hematoxylin and eosin staining), image analysis, epithelial immunoassay (EPISPOT), enzyme analysis, gene expression profiling analysis, therapeutic efficacy testing, concentrated cell culture, and therapeutic use of concentrated rare cells. In some embodiments, deficient plasma proteins and leukocytes can also be optionally recovered and subjected to other analyses such as inflammation studies and gene expression profiling.

[0140] In certain embodiments, cytoplasmic tumor cells (CTCs) collected from blood can be stained to identify them as potential tumor cells rather than blood cells. This allows for morphological identification of CTCs using colorimetric staining. Other methods are based on fluorescence staining. Several typical fluorescence staining methods for identifying cells as tumor cells use DAPI to identify the nucleus and fluorescent dye-conjugated cytokeratins 8, 18, and 19 to identify them as epithelial cells. Since normal epithelial cells are not found in blood, epithelial cells found in blood are accepted as tumor cells. CD45 antibodies are used to identify leukocytes by removing blood cells such as CTCs that are retained on a microfilter. Cells from blood that are DAPI-positive, CK8, 18, and 19-positive, and CD45-negative are commonly accepted as CTCs. Other markers such as EpCAM, MUC-1, and others are included to further provide increased specificity and fluorescence signaling.

[0141] In certain embodiments, captured CTCs are stained to specifically identify the origin of the tumor cells, such as those from the breast, prostate, or colon. For example, the cell marker PSA would identify its origin as prostate cancer. For each type of cancer, specific markers may be found either on the surface or within the cell.

[0142] In certain embodiments, the captured CTCs may be characterized by determining whether they contain specific mutations in the DNA. This can be identified by DNA, mRNA, microRNA expression by PCR, sequencing, or by antibodies, ligands, aptamers, and other substances that recognize the mutant protein.

[0143] In certain embodiments, captured CTCs may be characterized by determining the overexpression of a gene. Sometimes, the copy number of a gene is greater than necessary for each cell. When more copies of a gene are present, more copies of mRNA are produced, which in turn produces more copies of the protein. The copy number of a gene can be determined by FISH or ISH, and the amount of mRNA produced can be obtained by PCR, mRNA_FISH, and mRNA_ISH. The amount of protein produced can be determined by immunostaining for that protein. Cells overexpressing a marker will stain brighter for that marker than normal tissue. An example of overexpression is HER-2 in some subtypes of breast cancer.

[0144] In certain embodiments, captured CTCs can be characterized to determine whether they are living cells.

[0145] In certain embodiments, captured CTCs can be determined to be viable if they are viable. Viability can be determined by trypan blue staining or culture.

[0146] In certain embodiments, captured CTCs can be determined to be stem cells. They can be identified by their stem cell marker phenotype (CD44). + / CD44 - / flow or CK19 + / MUC-1 - It can be dyed for the purpose of ( ).

[0147] In certain embodiments, live CTCs may be captured by microfilters coated with analyte recognition elements such as antibodies, ligands, or aptamers. The analyte of interest is secreted by the CTCs. If the analyte is produced by the CTCs, secondary analyte recognition elements can be used to generate a detectable signal. This signal may be generated by a fluorescent dye. The concept is similar to EPISPOT.

[0148] In certain embodiments, live CTCs can be cultured directly on a microfilter to increase their number and to evaluate their properties. In other embodiments, CTCs can be backwashed from the microfilter before culture or sorting.

[0149] In other embodiments, microfilters formed from layers of photodestructible dry epoxy films according to embodiments of the present invention can be used in therapeutic applications to remove circulating tumor cells from the blood of cancer patients. Circulating tumor cells are the cause of cancer that spreads from its original site to other locations such as the brain, lungs, and liver. Most cancer patients die from metastatic cancer. In certain embodiments, microfiltration using microfilters formed according to embodiments of the present invention is a preferred method for removing circulating tumor cells from a patient's bloodstream because, when used to filter blood, it has a fast filtration rate, the microfilter does not retain white blood cells, and has very few red blood cells.

[0150] Microfiltration for circulating tumor cells in the blood can provide a wide array of diagnostic, predictive diagnostic, and research applications. To collect circulating tumor cells, previous research reports have utilized track-etch filters with randomly placed pores, where some pores are not straight, and microfilters with neatly arranged pores produced by reactive ion etching. In specific embodiments of the present invention, precisely pore-arranged microfilters formed from an epoxy-based photodestructible dry film are used to collect circulating tumor cells in the blood.

[0151] One exemplary application of a microfilter formed according to embodiments of the present invention is to monitor the effectiveness of a treatment by counting the number of circulating tumor cells (CTCs) collected in the blood. A high number of CTCs per milliliter of blood may indicate a short lifespan. Changes in the number of CTCs per milliliter of blood may indicate whether or not the treatment is working. A decrease in the number of CTCs indicates that the treatment is effective. Conversely, an increase in the number of CTCs indicates that the treatment is ineffective.

[0152] One exemplary use of a microfilter formed according to embodiments of the present invention is to use the microfilter to capture cells and subsequently culture the cells in a filter holder, or to culture cells after backwashing them from the microfilter. Various drugs to which cultured CTCs can be applied can be used to evaluate the efficacy of the drugs in order to determine the best treatment for the patient.

[0153] One exemplary application of a microfilter formed according to embodiments of the present invention is to determine gene mutations in CTCs in order to determine a suitable drug.

[0154] One exemplary use of a microfilter formed according to embodiments of the present invention is to determine the expression of a marker when a drug for treating a tumor by expression is present.

[0155] One exemplary application of a microfilter formed according to an embodiment of the present invention is to determine whether cancer is returning after reduction. If the number of CTCs in 7.5 ml of blood is greater than 5 and the CTC count is increasing over time, then the cancer is returning.

[0156] Another exemplary application of the microfilters manufactured according to embodiments of the present invention is the capture of circulating endothelial cells. Endothelial cells in peripheral blood provide information about various medical conditions.

[0157] Another exemplary application of the microfilters manufactured according to embodiments of the present invention is the capture of circulating fetal cells in the maternal blood during 11–12 weeks of gestation. Such fetal cells may include immature fetal nucleated erythrocytes. Fetal cells circulating in the peripheral blood of pregnant women can be used as targets in non-invasive genetic analysis. Fetal cells include epithelial (trophoblast) cells with a diameter of 14–60 μm, which are larger than peripheral blood leukocytes. In non-invasive prenatal diagnosis of hereditary disorders using PCR analysis of DNA targets or fluorescence in-situ hybridization (FISH) analysis of genes, high concentrations of circulating fetal cells may be used before performing genetic diagnosis.

[0158] Other exemplary uses of the microfilters manufactured according to embodiments of the present invention include collecting or concentrating stromal cells, mesenchymal cells, endothelial cells, epithelial cells, stem cells, non-hematopoietic cells, etc., from blood samples, collecting tumor or pathogenic cells from urine, and collecting tumor cells from cerebrospinal fluid and cerebrospinal fluid. Another exemplary use is collecting tumor cells from cerebrospinal fluid using the microfilter. Another exemplary use is capturing antigens originating from specimens or particle aggregates that bind to latex beads using the microfilter, so that the specimens or aggregated clusters coated on the beads are captured on the membrane surface.

[0159] Another exemplary use of a microfilter formed according to embodiments of the present invention is in the erythrocyte deformability test. Erythrocytes are highly flexible cells that easily deform to pass through pores. In some diseases, such as sickle cell anemia, diabetes, sepsis, or certain cardiovascular diseases, these cells become rigid and can no longer pass through small pores. Healthy erythrocytes are typically 7.5 μm in diameter and easily pass through a membrane with 3 μm pores, whereas cells with any of these conditions cannot. In the deformability test, a microfilter with a 5 μm aperture is used as a screening barrier. A blood sample is injected, and the membrane is positioned so that a constant pressure is applied. The filtration rate of the cells is then measured, and a decrease in filtration rate suggests a decrease in deformability.

[0160] Another exemplary use of microfilters formed according to embodiments of the present invention is the separation of leukocytes / erythrocytes. Blood cell populations with high concentrations of leukocytes (white blood cells) are often desirable for research and therapeutic use. Typical leukocyte sources include whole peripheral blood, leukocytapheresis, or the products of component depletion, as well as other less common sources such as umbilical cord blood. Erythrocytes in the blood can be lysed. The blood is then passed through a microfilter with small pores to retain the leukocytes. Another exemplary application is the use of microfilters in chemotactic applications. The membrane is used to investigate the response of leukocytes to toxins and to identify the innate immunity of whole blood. Since immunity can be transferred, this analytical method is used in the development of vaccines and drugs related to leukocytes. Another exemplary application is the use of microfilters for hemofiltration and / or transfusion. In such applications, microfilters can be used to remove large embolus, platelet aggregates, and other debris.

[0161] Furthermore, according to the embodiments of the present invention described above, a high-precision array of micropores can be fabricated on a polymer resist roll. Such an array can be used in applications where wafer-sized microfilters are unsuitable. Examples of such applications include water filtration and kidney dialysis.

[0162] While various embodiments have been described above, it should be understood that these embodiments are illustrative and not limiting to the invention. It will be apparent to those skilled in the art that various modifications to the form and details are possible without departing from the spirit and scope of the invention. Thus, the breadth and scope of the invention should not be limited by any of the exemplary embodiments described above, but rather defined solely by the following claims and their equivalents. Therefore, the embodiments of the invention should be considered in all respects as illustrative and non-limiting. Furthermore, it will be understood that any of the features, components, elements, etc. described above may be realized together in relation to different exemplary embodiments. [Explanation of Symbols]

[0163] 100, 400, 500, 700 dry membrane 110, 410 Exposed dried film 120, 420, 520 microfilters 116 Non-polymerized part 118, 418, 518 patterns 122 Aperture 180, 580 circuit boards 181 Polyimide film 182 Separator 197 Mask portion 198 Mask Patterns 199, 499, 599, 899 masks 416 Exposed area 422, 522 holes 497 Transparent part 498 Opaque area 501 Structure 516 parts 590, 690 support 597 Transparent part 598 patterns 660, 662 conductive layer 664 Insulator 665 Voltage 692 Water-cooled frame 693 Duct 702 rolls 770, 774, 775 Laura 772 Arrow 782 circuit boards 785 Structure 787 Working part 791, 891 Support 860 Clamp 887 Laminate 910, 912, 914, 916, 1010, 1012, 1014, 1016, 1018 Microfilters 920, 922, 924, 926, 928, 1020, 1022, 1024, 1028, 1029 holes 1030, 1032, 1034, 1036, 1038 Thickness Width 1040, 1042, 1044, 1045, 1046, 1047, 1048, 1049 1050, 1053, 1054 First surface 1052, 1056, 1057 Second surface 1210, 1212 dry membrane 1218 patterns 1222, 1232, 1242 Trench 1230, 1240 microfilters 1270, 1420 multilayer microfilters 1272 First surface 1274 Second surface 1280 Nonlinear passage 1282 First Aperture 1284 Second Aperture 1290 masks 1293 Mask Pattern 1295 Mask portion 1510 Precision filtration structure 1520 Microfilter 1522 hole 1530 Support Structure 1600 Coating 1655, 1672 microfilters 2500, 2540, 2561, 2563, 2580 equipment 2501, 2502 Side view 2541 Microfilter 2542 holes 2544 Wells 2551 Upper strip 2552 Bottom layer 2553 slots 2554 hole 2555 Upper Channel 2572 Base support 2573 hole 2575 posts 2581 Bottom layer 2582 Top Floor 2583 Well 3000 filter holder 3010 Entrance Unit 3020 Nut 3030 Outlet Unit 3050 Inlet Adapter 3100, 3220 filter holder 3110 Gasket 3120, 3320 microfilters 3200, 3300 Inlet container 3210, 3310 opening 3410, 3510 syringe 3420 Input container 3500, 3600 filtration system 3520 Plunger 3610 Syringe Pump 3620 Pusher Block 3710, 3810 Vacutainer Holder 3720 Input container 3730, 3830 filter holder 3740, 3840 Vacutainer 3800 filtration equipment 3820 Inlet Adapter 3850 needle

Claims

1. An inlet unit including a first volume and a first inlet unit opening to the first volume, A filter structure comprising a single polymer filter layer having a uniform thickness formed from a negative-type photodestructible dry film, the polymer filter layer having a plurality of apertures extending through the single polymer filter layer, and the filter structure being flexible so as to be processed on a roll or non-roll. An outlet unit including a second volume for housing the filter structure configured to be removable inside, and a second opening to the second volume, The first inlet unit opening and the second opening are located at opposite ends when the inlet unit is fixed to the outlet unit, and the filter structure is exposed for observing the filtered material collected on the filter structure and for performing an assay on the filtered material in the second volume through the first inlet unit opening. Includes a nut that is removably attached to the outlet unit and configured to removably secure the inlet unit to the outlet unit, The first inlet unit opening is exposed through the nut. Filtration device.

2. The filtration apparatus according to claim 1, further comprising an upper layer covering at least a portion of the first volume facing the first filter surface.

3. The above upper layer includes at least one first upper layer opening, The first upper layer opening described above is connected to the first entrance unit opening, The substance enters the first volume through the first upper layer opening. The filtration apparatus according to claim 2, wherein the substance enters the opening of the first inlet unit and passes from the first volume through the upper layer to the second volume so that it is collected on the filter structure.

4. The filtration apparatus according to claim 1, wherein the photoselectable dry film includes an epoxy resin-based photoselectable dry film.

5. The filtration apparatus according to claim 1, wherein the first filter surface of the filter structure includes a coating formed from a metallic substance, a nanoparticle colloidal substance, a chemical substance, or an organic substance.

6. The filtration apparatus according to claim 5, wherein the coating includes an analyte recognition element.

7. The filtration apparatus according to claim 1, wherein the filter structure includes a plurality of polymer filter layers, and the plurality of polymer filter layers form a laminate.

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