Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D29/00—Filters with filtering elements stationary during filtration, e.g. pressure or suction filters, not covered by groups B01D24/00 - B01D27/00; Filtering elements therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D39/00—Filtering material for liquid or gaseous fluids
  • B01D39/14—Other self-supporting filtering material ; Other filtering material
  • B01D39/16—Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D39/00—Filtering material for liquid or gaseous fluids
  • B01D39/14—Other self-supporting filtering material ; Other filtering material
  • B01D39/16—Other self-supporting filtering material ; Other filtering material of organic material, e.g. synthetic fibres
  • B01D39/1692—Other shaped material, e.g. perforated or porous sheets
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/14—Ultrafiltration; Microfiltration
  • B01D61/18—Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
  • B01D63/08—Flat membrane modules
  • B01D63/088—Microfluidic devices comprising semi-permeable flat membranes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/56—Labware specially adapted for transferring fluids
  • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
  • B01L3/5635—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors connecting two containers face to face, e.g. comprising a filter
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0681—Filter
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
  • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T29/00—Metal working
  • Y10T29/49—Method of mechanical manufacture
  • Y10T29/49826—Assembling or joining
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T29/00—Metal working
  • Y10T29/49—Method of mechanical manufacture
  • Y10T29/49826—Assembling or joining
  • Y10T29/49947—Assembling or joining by applying separate fastener
  • Y10T29/49963—Threaded fastener

Definitions

• the present invention relates generally to microfiltration devices, that contain polymer microfilters, methods of manufacturing the same, methods to use the microfiltration device, and applications of the devices.
• Some medical conditions may be diagnosed by detecting the presence of certain types of cells in bodily fluid.
• cells indicative or characteristic of certain medical conditions may be larger and/or less flexible than other cells found in certain bodily fluids. Accordingly, by collecting such larger and/or less flexible cells from a liquid sample of a bodily fluid, it may be possible to diagnose a medical condition based on the cells collected.
• Cells larger and/or less flexible than other cells present in a bodily fluid may be collected by filtering the bodily fluid.
• targeted cells indicative of a condition may be collected by passing a bodily fluid through a filter having openings that are too small for the target cells to pass through, but large enough for other cells to pass through.
• any number of analyses of the target cells may be performed. Such analyses may include, for example, identifying, counting, characterizing, and/or culturing the collected cells.
• microfilters It is desirable for microfilters to have precise pore dimensions, not break during use, and not be autofluorescent for fluorescent microscope imaging.
• microfilters are installed in a microfiltration device and liquid samples processed to collect cells based on size on the microfilter.
• a microfilter comprises a polymer layer formed from epoxy-based photo-definable dry film, and a plurality of apertures each extending through the polymer layer.
• a microfilter comprises a first polymer layer formed from epoxy-based photo -definable dry film and having a first aperture extending therethrough, and a second polymer layer formed from epoxy-based photo-definable dry film and having a second aperture extending therethrough, wherein the first and second apertures at least partially define a non-linear passage extending through the first and second layers.
• a method of manufacturing a microfilter comprises providing a first layer of epoxy-based photo-definable dry film disposed on a substrate, exposing the first layer to energy through a mask to form a pattern, defined by the mask, in the first layer of dry film, forming, from the exposed first layer of dry film, a polymer layer having a plurality of apertures extending therethrough, the plurality of apertures having a distribution defined by the pattern, and removing the polymer layer from the substrate.
• a method of forming a multi-layer microfilter comprises forming a first polymer layer comprising a plurality of first apertures from a first layer of epoxy-based photo-definable dry film disposed on a substrate, laminating a second layer of epoxy-based photo-definable dry film on the first polymer layer, and forming a second polymer layer comprising a plurality of second apertures from the second layer of dry film.
• filter holders to hold the microfilter are disclosed.
• filter holders are designed to keep the filter flat and fixed in place, allowing the implementation of various assay steps with ease.
• a method of using a microfilter comprises passing a liquid through a plurality of apertures of a microfilter formed from an epoxy-based photo-definable dry film, wherein the microfilter has sufficient strength and flexibility to filter the liquid, and wherein the apertures are sized to allow passage of a first type of bodily fluid cell and to prevent passage of a second type of bodily fluid cell.
• An exemplary implementation includes application of a negative pressure using either vacuum or filter holder connect to syringe(s), which can be performed manually, semi-manually using a syringe pump, or automated.
• filters can be coated with analyte recognition reagents to capture cells of interest from the body fluids.
• methods to collect liquid sample and shipping the filtered liquid sample using the microfilter inside the filter holder are also described.
• exemplary embodiments of the present invention provide methods to isolate cells, methods to recover the cells from the microfilter by backwashing using the filter holder, and methods to perform assay inside the filter holder.
• FIGS. 1A-1E are cross-sectional views illustrating multiple stages in a process for manufacturing a microfilter in accordance with exemplary embodiments of the present invention
• FIG. 2A is a flowchart illustrating a process for manufacturing a microfilter in accordance with exemplary embodiments of the present invention
• FIG. 2B is a flowchart illustrating a process for forming a microfilter from an exposed dry film in the process illustrated in FIG. 2A, in accordance with exemplary embodiments of the present invention
• FIGS. 3A-3E are cross-sectional views illustrating multiple stages in a process for manufacturing a microfilter 120 in accordance with exemplary embodiments of the present invention.
• FIGS. 4A-4D are cross- sectional views illustrating multiple stages in a process for manufacturing a microfilter in accordance with exemplary embodiments of the present invention
• FIGS. 5A-5D are cross- sectional views illustrating multiple stages in a process for manufacturing a plurality of microfilters from a plurality of layers of epoxy-based photo- definable dry film in accordance with exemplary embodiments of the present invention
• FIGS. 6A and 6B are cross-sectional views illustrating multiple stages in a process for attaching dry film structures to a support using an electrostatic chuck apparatus in a process for forming microfilters in accordance with exemplary embodiments of the present invention
• FIGS. 7 A and 7B are cross-sectional views illustrating multiple stages in a process for manufacturing microfilters from a roll of a dry film structure in accordance with exemplary embodiments of the present invention
• FIGS. 8 A and 8B are cross-sectional views illustrating multiple stages in a process for manufacturing microfilters from a plurality of rolls of dry film structures in accordance with exemplary embodiments of the present invention
• FIGS. 9A-9D are partial top views illustrating various microfilters aperture distributions in accordance with exemplary embodiments of the present invention.
• FIGS. 10A-10D are cross-sectional views illustrating microfilters having various thicknesses and various aperture shapes, sizes and distributions in accordance with exemplary embodiments of the present invention.
• FIG. 11 is a flow chart illustrating a process 1300 for manufacturing a multi-layer microfilter in accordance with exemplary embodiments of the present invention.
• FIGS. 12A-12K are cross-sectional views illustrating multiple stages in a process for manufacturing a multi-layer microfilter in accordance with exemplary embodiments of the present invention.
• FIG. 12L is a top view of multi-layer microfilter in accordance with exemplary embodiments of the present invention.
• FIGS. 13A and 13B are top views illustrating multiple stages in the process of FIG. 11;
• FIGS. 14A and 14B are cross-sectional views of a multi-layer microfilter 1420 in accordance with exemplary embodiments of the present invention.
• FIG. 14C is a top view of a multi-layer microfilter 1420 of FIGS. 14A and 14B;
• FIG. 15 is a cross-sectional view of a microfiltration structure including a microfilter and a support structure in accordance with exemplary embodiments of the present invention.
• FIG. 16A is a cross-sectional view of a microfiltration structure including pores in the microfilter and posts above the microfilter in accordance with exemplary embodiments of the present invention.
• FIG. 16B is a top view of a microfiltration structure including pores in the microfilter and posts above the microfilter in accordance with exemplary embodiments of the present invention.
• FIG. 16C is a top view of a microfiltration structure including rectangular pores in the microfilter and posts above the microfilter in accordance with exemplary embodiments of the present invention.
• FIGS. 16D and 16E are side views of another microfilter according to an exemplary embodiment of the present invention made by two layers of material with different patterns in each layer.
• FIG. 16F is the top view of the microfilter according to an exemplary embodiment of the present invention made by two layers of material with different patterns in each layer.
• FIGS. 16G and 16H are side views of another microfilter according to an exemplary embodiment of the present invention made by two layers of material with different patterns in each layer.
• FIGS. 161 and 16J are side views of a device according to an exemplary embodiment of the present invention with many wells suitable for many assays made by two layers of material with opening on the top layer and solid bottom layer.
• FIG. 17 is a cross-sectional views of coated planar microfilters in accordance with exemplary embodiments of the present invention.
• FIGS. 18 is a cross-sectional views of coated structured microfilters in accordance with exemplary embodiments of the present invention.
• FIG. 19 illustrate an example of lithographically fabricated microfilter according to an exemplary embodiment of the present invention using epoxy -based photo-definable dry film with the pore geometry and locations defined by an optical mask;
• FIGS. 20A-20D illustrates the components of a filter holder in accordance with exemplary embodiments of the present invention
• FIGS. 21A-21E illustrates the assembly process of the microfilter in the filter holder illustrated in FIGS. 20A-20D in accordance with exemplary embodiments of the present invention
• FIGS. 22A-22B illustrate input container options for input liquid sample and reagents to be attached to the assembled sample holder illustrated in FIG. 21E in accordance with exemplary embodiments of the present invention
• FIG. 23 is an illustration of forming an input container according to an exemplary embodiment of the present invention to be attached to the assembled sample holder illustrated in FIG. 21E using a syringe with Luer lock without the plunger and an adaptor in accordance with exemplary embodiments of the present invention;
• FIG. 24 illustration of the filtration system according to an exemplary embodiment of the present invention to perform the assays incorporating a microfilter inside the filter holder in accordance with exemplary embodiments of the present invention
• FIG. 25 illustrates the filtration system according to an exemplary embodiment of the present invention to perform some steps of assays with the input container removed in accordance with exemplary embodiments of the present invention
• FIG. 26 illustrates the filtration system according to an exemplary embodiment of the present invention using filtration system illustrated in FIG. 24, where the outlet syringe is pulled by a syringe pump.
• FIGS. 27A-27B shows the configuration according to an exemplary embodiment of the present invention using vacutainer holder to draw the liquid sample from the input sample holder through the filter to a vacutainer.
• FIGS. 28A-28B shows the configuration according to an exemplary embodiment of the present invention using vacutainer holder to draw the liquid sample from the patient through the filter to a vacutainer. This can be used, for example, to filter circulating tumor cells at time of blood drawn in accordance with embodiments of the present invention;
• FIGS. 29A-29B are flowcharts illustrating the options in procedures of collecting liquid samples and testing liquid samples in accordance with exemplary embodiments of the present invention.
• FIG. 29C is a flowchart illustrating the options to filter rare cells when collecting blood using vacutainers in accordance with exemplary embodiments of the present invention.
• FIG. 30A is a flowchart illustrating a filtration process using a microfilter in accordance with exemplary embodiments of the present invention.
• FIG. 30B is a flowchart illustrating a filtration process using a microfilter in accordance with exemplary embodiments of the present invention.
• FIG. 30C is a flowchart illustrating the process of performing some types of the assays of the cells collected on the microfilter in the filter holder in accordance with exemplary embodiments of the present invention
• aspects of the present invention are generally directed to a microfilter comprising a polymer layer formed from an epoxy-based photo-definable dry film.
• the microfilter includes a plurality of apertures each extending through the polymer layer.
• the microfilter may be formed by exposing the dry film to energy through a mask and developing the exposed dry film.
• the dry film may be exposed to energy in the form of ultraviolet (UV) light.
• the dry film may be exposed to energy in the form of X-rays.
• the polymer layer has sufficient strength and flexibility to filter liquid.
• the apertures are sized to allow passage of a first type of bodily fluid cell and to prevent passage of a second type of bodily fluid cell.
• the microfilter may be used to perform assays on bodily fluids.
• the microfilter may be used to isolate and detect large rare cells from a bodily fluid.
• the microfilter may be used to collect circulating tumor cells (CTCs) from peripheral blood from cancer patients passed through the microfilter.
• CTCs circulating tumor cells
• the microfilter may be used to collect circulating endothelial cells, fetal cells and other large cells from the blood and body fluids.
• the microfilter may be used to collect large cells from processed tissue samples, such as bone marrows.
• cells collected using the microfilter may be used in downstream processes such as cell identification, enumeration, characterization, culturing, etc.
• multiple layers of epoxy-based photo- definable dry film may be exposed to energy simultaneously for scaled production of microfilters.
• a stack of epoxy-based photo-definable dry film layers is provided, and all of the dry film layers in the stack are exposed to energy simultaneously.
• a dry film structure including epoxy-based photo-definable dry film disposed on a substrate is provided in the form of a roll. In such embodiments, a portion of the structure may be unrolled for exposure of the dry film to energy. In certain embodiments, portions of a plurality of rolls may be exposed to energy simultaneously.
• FIGS. 1A-1E are cross-sectional views illustrating multiple stages in a process for manufacturing a microfilter 120 in accordance with embodiments of the present invention.
• FIG. 2A is a flowchart illustrating a process 200 for manufacturing a microfilter in accordance with embodiments of the present invention. The exemplary process of FIG. 2A will be described below with reference to FIGS. 1A-1E. Other embodiments of the process illustrated in FIG. 2A will be described below with reference to FIGS. 3A-8B.
• a layer of epoxy-based photo-definable dry film 100 (which may be referred to herein as "dry film 100") disposed on a substrate 180 is provided.
• dry film 100 is laminated on substrate 180 at block 220.
• a silicon wafer is coated with a thin layer of metallic material, such as copper, and dry film 100 is laminated on the metallic material at block 220.
• a dry film 100 with a substrate 180 already attached may be obtained and provided at block 220.
• an "epoxy-based photo-definable" substance refers to a substance including or formed from a photo-definable epoxy resin, such as a polyfunctional epoxy resin, bisphenol A epoxy resin, epoxidized polyfunctional bisphenol A formaldehyde novolac resin, etc. Examples of photo- definable epoxy resins may be found in U.S. Patent Nos.
• an "epoxy-based photo-definable dry film” is a dry film including or formed from an epoxy-based photo-definable substance.
• Examples of epoxy-based photo-definable dry films that may be used in accordance with embodiments of the present invention may be found in U.S. Patent Nos. 7,449,280, 6,391,523, and 6,558,868, and U.S. Patent Publication Nos. 2010/0068648 and 2010/0068649. Formulations of epoxy-based photo-definable dry films are not limit to those described here.
• liquid resist form of the epoxy-based photo-definable dry films before they are coated on substrate can be spin coated on substrate and dried to obtain the dry film on the substrate.
• substrate 180 is a thin copper foil.
• smooth substrates are preferable because irregularities in the surface of the substrate to which the dry film is laminated are transferred to a surface of the dry film.
• a thin copper film is preferred as a substrate so that the substrate may be removed in a relatively short amount of time.
• substrate 180 may be a silicon wafer, a polyimide film such as Kapton, or any other suitable material.
• dry film 100 is exposed to energy through a mask 199 to form an exposed dry film 110 at block 240.
• dry film 100 is exposed to energy in the form of ultraviolet (UV) light through an optical mask 199 having a mask portion 197 that is transparent to UV light and a mask pattern 198 formed from a thin film of material that is opaque to UV light.
• UV ultraviolet
• dry film 100 may be exposed to X-rays through an X-ray mask at block 240, instead of being exposed to UV light through optical mask 199.
• epoxy-based photo definable dry film 100 is a negative resist.
• a "negative resist” is a photo-definable substance that becomes polymerized when exposed to certain kinds of energy, such as UV light or X-rays. Examples of negative resist epoxy-based photo-definable dry films that may be used in accordance with embodiments of the present invention.
• the portions of exposed dry film 110 that were exposed to UV light through mask 199 become polymerized, leaving portions 116 that are not polymerized.
• the polymerized and non-polymerized portions of exposed dry film 100 form a pattern 118 that is defined by optical mask 199.
• pattern 118 of exposed dry film 110 is defined by a pattern 198 of optical mask 199, where pattern 198 is formed by material opaque to UV light.
• pattern 198 may be formed by a thin film of material opaque to UV light, such as a thin film of chromium.
• a positive epoxy-based photo-definable dry film may be used instead of a negative dry film.
• the process for forming a microfilter from the positive dry film is similar to the process for forming a microfilter described in relation to FIGS. 1A-2A, except that a different mask may be used, as described below in relation to FIGS. 4A-4D.
• a "positive resist” is a photo-definable substance in which polymeric bonds are broken when the substance is exposed to certain kinds of energy, such as UV light or X-rays.
• the positive resist may be a resist based on polydimethylglutarimide (such as PMGI, LOR available from MicroChem), an acetate and xylene free resist (such as an S I 800® series resist available from Shipley Corp.), or another type of positive resist.
• polydimethylglutarimide such as PMGI, LOR available from MicroChem
• an acetate and xylene free resist such as an S I 800® series resist available from Shipley Corp.
• positive resist layers that are greater than a few microns in thickness, negative resists are generally much 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 polymethylmethacrylate (PMMA), and a synthetic polymer of methyl methacrylate.
• PMMA polymethylmethacrylate
• Other examples of positive resists are acrylics, polyimide, polyesters, such as polyethylene terephthalate (PET) (MYLARTM), etc.
• a microfilter may be formed from a photo-definable dry film that is not epoxy based, in accordance with embodiments of the present invention.
• the dry film may be a positive or a negative resist.
• a microfilter may be formed from a photo-definable liquid resist, rather than a dry film.
• the photo-definable liquid resist may be a positive resist or a negative resist.
• the photo -definable liquid resist may be liquid polyimide.
• the photo-definable liquid polyimide may be positive resist or negative resist. The liquid resist is spin coated on the substrate and dried to form the dry film on the substrate.
• a microfilter 120 having a plurality of apertures 122 extending through the microfilter is formed from exposed dry film 110.
• microfilter 120 includes a polymer layer formed from epoxy-based photo-definable dry film and a plurality of apertures extending through the polymer layer.
• a microfilter includes one or more polymer layers and one or more apertures extending though each of the one or more polymer layers.
• an "aperture” refers to any type of passage, pore, trench, gap, hole, etc., that extends between outer surfaces of a layer or other structure.
• apertures 122 are pores 122.
• exposed dry film 110 is developed to remove non-polymerized portions 116 to form microfilter 120 having pores 122.
• exposed dry film 110 is developed by applying a developer to dry film 110 to dissolve non-polymerized portions 116.
• the developer is an aqueous solution that dissolves non-polymerized portions 116 when exposed dry film 110 is submerged in the developer.
• microfilter 120 having pores 122 is removed from substrate 180 to form a free-standing microfilter 120, as shown in FIG. IE.
• a microfilter is a structure of one or more polymer layers including one or more apertures extending between outer surfaces of the structure, wherein the structure has sufficient strength and flexibility to filter a liquid passed through the one or more apertures.
• a microfilter may include apertures having dimensions small enough to prevent one or more types of bodily fluid cells from passing through the apertures when a bodily fluid or liquid containing a bodily fluid is passed through the filter, wherein the dimensions of the apertures are also small enough to prevent one or more other types of bodily fluid cells from passing through the filter.
• a microfilter refers to any cell that may be found in a bodily fluid of a patient, such as red or white blood cells, large rare cells, such as CTCs and fetal cells, circulating endothelial cells, etc.
• a microfilter includes apertures sized to allow passage of a significant number of red blood cells and to prevent passage of a significant number of CTCs.
• a microfilter formed from one or more layers of epoxy-based photo-definable dry film may be a polymeric microfilter.
• substrate 180 may be copper foil.
• copper substrate 180 may be removed from microfilter 120 using nitric acid, ferric chloride or another well-known reagent in one variation of block 280.
• the reagent may be used to etch away copper substrate 180 in order to remove it from microfilter 120.
• substrate 180 may be another type of metallic foil, such as aluminum, and may be removed at block 280 by well-known methods.
• FIG. 2B is a flowchart illustrating a process for forming a microfilter from an exposed dry film at block 260 of FIG. 2A in accordance with embodiments of the present invention.
• the process 260 for forming the microfilter comprises forming, from the exposed dry film, a polymer layer comprising a plurality of apertures.
• a post bake process is performed on exposed dry film 110 disposed on substrate 180 at block 262.
• the post bake process includes exposing dry film 110 to a relatively high temperature to post bake dry film 110.
• dry film 110 is developed by applying a developer to dry film 110, as described above in relation to FIGS. 1A-1E.
• a hard bake process is performed on the developed dry film 110.
• the hard bake process includes exposing dry film 110 to a relatively high temperature.
• the hard bake process of block 266 may be omitted.
• microfilter 120 is formed by post baking the exposed dry film 110 at block 262, and developing dry film 110 at block 264. The processes described above in relation to FIG. 2B may be used with any of the embodiments described herein.
• the process for forming a polymer layer of a microfilter from an epoxy-based photo-definable dry film may include exposing the dry film to energy, performing a post-bake process, developing the exposed dry film, and/or post baking the exposed dry film, as described above.
• FIGS. 3A-3E are cross-sectional views illustrating multiple stages in a process for manufacturing microfilter 120 in accordance with embodiments of the present invention.
• the substrate is a polyimide film 181.
• a layer of epoxy-based photo-definable dry film 100 disposed on polyimide film 181 is provided with a separator 182 disposed between a portion of dry film 100 and polyimide film 181.
• a separator 182 is disposed between a portion of dry film 100 and polyimide film 181 along an edge of dry film 100.
• separator 182 may be disposed along one or more edges of dry film 100, or at other locations between dry film 100 and polyimide film 181.
• Separator 182 may be formed from a polyimide film (such as a KAPTON film) or from any other suitable material that can be laminated to dry film 100 and withstand the temperature of a hard bake process.
• microfilter 120 is formed from exposed dry film 110 at blocks 240 and 260, as described above in relation to FIGS. IB- ID.
• microfilter 120 includes a polymer layer comprising a plurality of apertures there through.
• microfilter 120 is removed from polyimide film 181 by grasping an exposed end of separator 182 and using separator 182 to peel microfilter 120 from polyimide film 181.
• separator 182 is removed from dry film 100 to obtain a free-standing microfilter 120, as shown in FIG. 3E. Removing microfilter 120 from layer 181, and removing separator 182 from microfilter 120, are two steps performed in one variation of block 280, in accordance with embodiments of the present invention.
• a liquid resist may be used instead of dry film 100.
• a substrate is coated with a thin layer of a metallic substance and an epoxy-based liquid photoresist is spin coated onto the metallic substance to provide a layer of an epoxy-based photo-definable substance on a substrate, in one variation of block 210.
• the substrate may be a silicon wafer
• the metallic substance may be copper
• the liquid photoresist may be an epoxy-based photo- definable liquid.
• the epoxy-based photo-definable liquid is a liquid negative resist, such as SU-8.
• microfilter 120 is formed from the exposed layer at blocks 240 and 260, as described above in relation to FIGS. IB-ID.
• microfilter 120 is released from the substrate by etching away the metallic substance via conventional processes as described above.
• the liquid resist may be a liquid negative resist, such as SU-8 or KMPR® available from MicroChem Corp.
• a liquid negative resist may be used instead of dry film 100, and a positive resist between the negative resist and the substrate may be used as a release layer.
• a liquid positive resist is spin coated on a substrate, the positive resist is exposed to energy (such as UV light) at the appropriate dose for the thickness of the coating, and a liquid, epoxy-based negative resist is spin coated on the positive resist.
• the positive resist may be exposed to energy without the use of a mask.
• microfilter 120 is released from the substrate by developing the positive resist.
• the same developer may be used to develop both the positive and the negative resists.
• one developer may be used to form the pores in microfilter 120, and another developer may be used to release the microfilter from the substrate.
• 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 polymethylmethacrylate (PMMA), and a synthetic polymer of methyl methacrylate.
• a negative epoxy-based photo-definable dry film 100 may be used in combination with a positive resist release layer. Such embodiments are similar to the embodiments described above utilizing a positive resist release layer, except that a layer of the negative dry film 100 may be laminated on the spin coated positive resist at block 210, rather than spin coating a liquid negative resist.
• FIGS. 4A-4D are cross- sectional views illustrating multiple stages in a process for manufacturing a microfilter 420 in accordance with embodiments of the present invention. In the embodiment illustrated in FIGS.
• a layer of positive, epoxy-based photo-definable dry film 400 (which may be referred to herein as "dry film 400") disposed on substrate 180 is provided at block 220.
• positive dry film 400 is laminated on substrate 180 at block 220.
• positive dry film 400 is exposed to energy at block 240, as described above in relation to FIG. IB and 1C, except that, rather than exposed portions of dry film 400 becoming polymerized, polymeric bonds of dry film 400 are broken at portions 416, which are exposed to energy (e.g., UV light) through mask 499.
• a pattern 418 of exposed portions 416 and unexposed portions is formed in exposed dry film 410. As shown in FIG.
• mask 499 includes a transparent portion 497 and an opaque portion 498.
• mask 199 of FIG. IB is used with a negative resist and is configured to cover portions of dry film 100 where pores will be formed in dry film 100.
• opaque portions 498 of mask 499 are configured to cover all portions of positive dry film 400 except the locations where apertures will be formed, allowing UV light to pass through mask 499 to positive dry film 400 at locations where apertures are to be formed in positive dry film 400.
• a microfilter 420 is formed from exposed dry film 410 in one variation of block 260, by developing dry film 410 using a developer that dissolves the portions 416 of dry film 400 where the polymeric bonds were broken.
• microfilter 420 having apertures 422 is removed from substrate 180 to form a free-standing polymeric microfilter 420, as shown in FIG. 4D.
• microfilter 420 includes a polymer layer formed from epoxy-based photo-definable dry film and includes a plurality of apertures extending through the polymer layer.
• the apertures 422 are pores 422.
• microfilter 420 may be released from the substrate by developing the positive resist, as described above.
• FIGS. 5A-5D are cross- sectional views illustrating multiple stages in a process for manufacturing a plurality of microfilters from a plurality of layers of epoxy-based photo- definable dry film in accordance with embodiments of the present invention.
• a plurality of dry film structures 501 each including a layer of epoxy-based photo-definable dry film 500 (which may be referred to herein as "dry film 500") disposed on a substrate 580, are provided at block 220.
• dry films 500 disposed on substrates 580 are provided at block 220 by stacking structures 501 on a support 590, as illustrated in FIG. 5A.
• dry films 500 in the stack of structures 501 are simultaneously exposed to energy in the form of X-rays through an X-ray mask 599 at block 240 of FIG. 2A.
• the penetration of X-rays is much deeper than UV light. Unlike UV light, X-rays do not diverge within a material having a thickness of less than 5 mm, even for features significantly smaller than one micron.
• X-ray lithography may be typically performed on a beamline of a synchrotron.
• X-ray lithography can be used for both negative and positive resists.
• dry films 500 are each negative resists.
• dry films 500 may be positive resists.
• a mask configured to form apertures in a positive resist, as described above in relation to mask 499 of FIG. 4B, may be used.
• dry films 500 may be may be attached to support 290 and stacked directly on one another, without each being disposed on a respective substrate.
• each dry film 500 exposed to X-rays through mask 599 become polymerized, leaving portions 516 of dry film 500 that are not polymerized.
• the polymerized and non-polymerized portions of each dry film 500 form a pattern 518 that is defined by pattern 598 of optical mask 599.
• mask 599 includes an X-ray transparent portion 597, and a pattern 598 configured to substantially block X-rays.
• pattern 598 is formed from gold.
• X-ray transparent portion 597 may be a thin graphite sheet or a silicon wafer. In the embodiment illustrated in FIGS.
• each of substrates 580 transmits most of the X-ray energy applied to it.
• substrates 580 are formed from a metallic foil. In such embodiments, when the foil is sufficiently thin, each substrate 580 will transmit most of the X- ray energy applied to it.
• the number of structures 501 that may be stacked and then exposed simultaneously is based on the reduction in the dose of the X-rays caused by the X-rays passing through the metallic foil.
• the plurality of exposed dry films 510 are developed to form a plurality of microfilters 520 each having apertures 522 in a manner similar to that described above in relation to FIGS. 1A-1E.
• apertures 522 are pores 522.
• the process illustrated in FIGS. 5B and 5C may be performed in one variation of block 260. In such embodiments, structures 501 are separated from one another, as shown in FIG. 5B, and a post bake procedure is performed on exposed dry films 510 disposed on respective substrates 580 in one variation of block 262.
• each of exposed dry films 510 is developed, as described above, in one variation of block 264 to form pores 522 in each of dry films 500, as shown in FIG. 5C.
• a hard bake procedure is performed on dry films 510 disposed on respective substrates 580, in one variation of block 266, to form microfilters 520 having pores 522.
• the hard bake procedure may be omitted.
• substrates 580 are chemically removed from microfilters 520, as described above, to obtain free-standing microfilters 520 having pores 522, as shown in FIG. 5D.
• each of microfilters 520 is a polymer layer including apertures 522.
• each of substrates 580 may be formed from a metallic foil.
• substrate 580 may be a polymer based substrate that transmits most of the X-rays applied to it, and which has a melting point higher than a post bake temperature for dry film 500.
• substrate 580 may be formed from a positive resist.
• substrate 580 may be exposed to energy, such as UV light or X-rays, sufficient to break polymeric bonds in the positive resist such that substrate 580 may be removed chemically by a developer solution at block 280 of FIG. 2A.
• substrate 580 may be a polyimide film and may be removed at block 280 by peeling the polyimide substrate 580 from microfilter 520.
• layers of epoxy-based photo-definable dry film 500 may be stacked and simultaneously exposed without each of the layers being disposed on a respective substrate.
• dry films 500 in one variation of block 220, dry films 500 are stacked on a support 590 without substrates disposed between adjacent dry films 500.
• the stacked dry films 500 are exposed in one variation of block 240.
• the process illustrated in FIG. 2B may be performed at block 260.
• the exposed dry films 510 may be separated and placed on separate substrates on which exposed dry films 510 undergo a post bake process in one variation of block 262.
• the substrates used are able to withstand the post bake temperature and can be dissolved by water or one or more chemicals. While attached to respective substrates, exposed dry films 510 are developed at block 264 and may undergo a hard bake procedure at block 266. At block 280, the substrates 580 are removed from the microfilters 520 formed from exposed dry films 510.
• structures 501 may be attached to support 590 using adhesive, a clamp, or any other suitable mechanism or method.
• structures 501 are held to a support by an electrostatic chuck.
• FIGS. 6A and 6B are cross-sectional views illustrating multiple stages in a process for attaching dry film structures 501 to a support using an electrostatic chuck apparatus 600 in a process for forming microfilters in accordance with embodiments of the present invention.
• a plurality of dry film structures 501 each including a layer of epoxy-based photo-definable dry film 500 disposed on a substrate 580, are provided stacked on a support 690, as shown in FIG. 6A.
• support 690 includes a water cooling frame 692 with a duct 693, an insulator 664 disposed on frame 692, and a conductive layer 662 disposed on insulator 664. Additionally, a transparent conductive layer 660 is placed on the stack of structures 501 such that the stack of structures 501 is disposed between conductive layers 660 and 662, as shown in FIG. 6A. Also as shown in FIG. 6A, a circuit connecting the conductive layers is open so that a voltage 665 of zero is applied to the conductive layers.
• FIG. 6B closing the circuit between the conductive layers and applying a non-zero voltage 665 between conductive layers 660 and 662 causes apparatus 600 to press together structures 501 between conductive layers 660 and 662.
• X-rays may be applied to dry films 500 through X-ray mask 599, as described above in relation to FIGS. 5A-5D.
• stacking structures 501 on apparatus 600 and pressing together structures 501 using apparatus 600, as described above in relation to FIGS. 6A and 6B may be performed in one variation of block 220 of FIG. 2A.
• FIGS. 7 A and 7B are cross-sectional views illustrating multiple stages in a process for manufacturing microfilters from a roll of a dry film structure in accordance with embodiments of the present invention. In the embodiment illustrated in FIGS.
• a dry film structure 785 is provided in the form of a roll 702 of the dry film structure 785.
• Dry film structure 785 includes a layer of epoxy-based photo-definable dry film 700 (which may be referred to herein as "dry film 700") disposed on a removable substrate 782.
• substrate 782 may be a chemically dissolvable metallic foil.
• the metallic foil may include aluminum or copper, which may be etched away as described above.
• a portion of roll 702 is disposed on a roller 774 and another portion is disposed on a roller 775.
• a working portion 787 of dry film structure 785 extends between rollers 774 and 775 and is held substantially flat by rollers 770 for exposure to energy through mask 199.
• a layer of epoxy-based photo-definable dry film 700 disposed on a substrate 782 is provided, in one variation of block 220, by unrolling a portion of dry film structure 785 from roll 702 and advancing the portion of structure 785 in the direction of arrow 772 to provide working portion 787 of structure 785 between support 791 and mask 199.
• the working portion 787 provided at block 220 includes a portion of dry film 700 that has not been patterned by exposure to energy through a mask.
• support 791 and mask 199 are moved away from structure 785 when structure 785 is advanced.
• mask 199 and support 791 are moved adjacent to structure 785, and dry film 700 is exposed to energy through mask 199, as shown in FIG. 7B, in one variation of block 240 of FIG. 2A.
• mask 199 is an optical mask and the energy is UV light, although a different type of energy may be used along with a different mask, as described above.
• dry film 700 is a negative resist.
• dry film 700 may be a positive resist.
• a mask configured to form pores in a positive resist, as described above in relation to mask 499 of FIG. 4B, may be used.
• Exposing dry film 700 to energy through the mask forms a pattern in dry film 700, as described above in relation to other embodiments.
• support 791 presses against structure 785, as shown in FIG. 7B, to stretch dry film 700 for the exposure process to thereby provide additional tension and stability to dry film 700 during the exposure process.
• structure 785 may be advanced again as described above, to provide a new working portion 787 that has not yet been exposed at block 220, and the new working portion 787 may be exposed at block 240, as described above.
• this process of advancing structure 785 and exposing dry film 700 may be continuously repeated. In some embodiments, the process may be repeated until most or all portions of dry film 700 have undergone an exposure process.
• a microfilter having apertures is formed from an exposed portion of dry film 700 by developing the exposed portion, as described above in relation to other embodiments, in one variation of block 260.
• the exposed portion of dry film 700 may be developed before it is rolled onto roller 775, or may be developed after all desired portions of dry film 700 have been exposed.
• the process illustrated in FIG. 2B may be performed at block 260.
• the exposed portion of dry film 700 may be advanced through an oven for a post bake procedure at block 262, the exposed portion of dry film 700 may be developed at block 264, and then undergo a hard bake procedure at block 266.
• the procedures at blocks 262, 264 and 266 may be performed after all desired portions of dry film 700 have been exposed.
• the hard bake procedure may be omitted.
• substrate 782 is removed at block 280, as described above in relation to other embodiments.
• individual microfilters are diced from the roll of dry film from which the microfilters were formed.
• forming microfilters from a dry film provided as a roll may simplify the manufacture of microfilters in accordance with embodiments of the present invention, and may allow automation of the manufacturing process.
• FIGS. 8 A and 8B are cross-sectional views illustrating multiple stages in a process for manufacturing microfilters from a plurality of rolls of dry film structures in accordance with embodiments of the present invention.
• the embodiment illustrated in FIGS. 8 A and 8B is similar to the embodiment illustrated in FIGS. 7A and 7B, except that layers of epoxy-based photo-definable dry film 700 of multiple rolls 702 are exposed to energy simultaneously.
• a plurality of layers of epoxy-based photo-definable dry film 700 each disposed on a respective substrate 782 are provided, in one variation of block 220, by advancing the structure 785 of each roll 702 in the direction of arrow 772 to provide a stack 887 of structures 785 between support 891 and mask 899.
• mask 899 and support 891 are moved adjacent to the stack 887, and the portions of dry films 700 in stack 887 disposed between mask 899 and support 891 are exposed to energy simultaneously through mask 899, as shown in FIG. 8B, in one variation of block 240 of FIG. 2A.
• dry films 700 are each negative resists.
• dry films 700 may be positive resists.
• a mask configured to form apertures in a positive resist, as described above in relation to mask 499 of FIG. 4B, may be used. Further processes for forming microfilters from dry films 700 are similar to the processes described above in relation to the embodiment illustrated in FIGS. 7 A and 7B.
• support 891 is disposed on a water cooling frame 692 including a duct 693.
• working portions 887 of structure 785 may be securely held in place between support 891 and mask 899 by a clamp 860.
• stack 887 may be held secure using an electrostatic chuck, as described above in relation to other embodiments.
• the number of dry films 700 exposed simultaneously may be determined based on the precision yielded when exposing a stack of a particular number of films.
• forming microfilters from a plurality of dry films provided as a plurality of rolls, as described above, may simplify the manufacture of microfilters and/or facilitate high volume production of microfilters.
• each roll 702 includes only the dry film and not any substrate.
• each roll 702 may include an additional cover layer on dry film 700.
• substrate 782 is disposed on a first side of dry film 700 and the cover layer on the opposite side of dry film 700.
• lithography-based microfabrication in accordance with embodiments of the present invention may enable efficient mass production of highly uniform precision microfilters.
• fabricating microfilters in accordance with embodiments of the present invention may yield increased porosity and pore uniformity in the microfilters produced.
• FIGS. 9A-9D are partial top views illustrating various microfilters aperture distributions in accordance with embodiments of the present invention.
• microfilters having different aperture sizes, shapes and distributions may be provided.
• certain combinations of aperture size, shape and distribution may be more advantageous than others for a particular application of a microfilter.
• a microfilter having round pores each having a diameter of 7-8 microns may be preferable in certain embodiments.
• a microfilter having round pores each with a diameter of 7- 8 microns can trap the rare cells while retaining a very small percentage of blood cells.
• microfilters 910 and 912 each have a uniform distribution of pores 920 and 922, respectively. Additionally, pores 920 are uniform in size, as are pores 922.
• microfilter 914 includes uniform rectangular pores 924, distributed over microfilter 914 in several groupings of pores 924.
• microfilter 916 a plurality of pores 926 of a first size, and a plurality of pores 928 of a second size. In other embodiments, any or pores 920, 922, 924, 926 and 928 may be any other type of aperture.
• microfilters 910, 912, 914 and 916 may be manufactured using any of the microfilter manufacturing processes described above in accordance with embodiments of the present invention. Additionally, any of the microfilter manufacturing processes described above in accordance with embodiments of the present invention may be used to form apertures of a plurality of different cross-sectional shapes. For example, in certain embodiments, apertures may be formed which have the cross-sectional shape of a circle, triangle, square, rectangle, ellipse, oval, trapezoid, parallelogram, etc.
• FIGS. 10A-10E are cross-sectional views illustrating microfilters having various thicknesses and various aperture shapes, sizes and distributions in accordance with embodiments of the present invention.
• FIGS. 10A and 10B show microfilters 1010 and 1012, each formed via one of the processes described above in accordance with embodiments of the present invention.
• Microfilter 1010 includes a plurality of pores 1020, each having a width 1040.
• Microfilter has a thickness 1030 substantially perpendicular to the width 1040 of pores 1020. In the embodiment illustrated in FIG. 10A, thickness 1030 is not significantly larger than width 1040.
• the thickness of the microfilter is on the same order as the width of one or more pores of the microfilter in order to reduce the pressure required to pass a liquid sample through the pores.
• the thickness of a microfilter is significantly greater than the width of some or all of the pores, a much larger amount of pressure may be applied to the microfilter to pass a liquid sample through a microfilter than if the microfilter has a thickness on the same order as some or all of the pores. Passing the liquid sample through the filter with a relatively large amount of pressure may distort the shape of one or more pores, or risk breaking the microfilter.
• a microfilter having a thickness of 8-14 microns may be preferable in certain embodiments.
• a microfilter for such an application may have pores each having a diameter of 7-8 microns and a thickness of 8-14 microns.
• a microfilter for such applications may include a rectangular aperture having a width of between 5- 7 microns and a length greater than 7 microns, wherein the length and width of the aperture are both substantially perpendicular to the thickness of the microfilter.
• the rectangular aperture may be an elongate trench.
• the width of the apertures in the microfilter are near in size to the thickness of the microfilter.
• the thickness of the microfilter is less than ten times the width of some or all the pores. In other embodiments, the thickness of the microfilter is within 10 microns of the width of some or all of the pores.
• Microfilters formed in accordance with embodiments of the present invention may be used in applications other than capturing circulating tumor cells from blood.
• the desired aperture geometry, aperture dimensions, aperture distribution, microfilter materials, microfilter thickness, microfilter size, etc. may vary for different applications.
• desired aperture geometry, dimensions, and distribution may be provided by using an appropriate mask, such as an optical or X-ray mask.
• a consideration for microfilters is strength of the material from which the microfilter is made to prevent breakage of the filter material or distortion of the aperture shape.
• microfilter 1012 has a plurality of pores 1022 each having a width 1042.
• Microfilter 1012 also has a thickness 1032 that is substantially perpendicular to the width 1042 of pores 1022.
• the thickness 1032 of microfilter 1012 is greater than the thickness of microfilter 1010.
• pores 1022 are uniform in size and are each substantially perpendicular to a first surface 1050 and a second surface 1052 of microfilter 1012.
• microfilter 1014 has pores 1024 with a first width 1044 and pores 1026 with a second width 1046 that is smaller than the first width 1044.
• Microfilter 1014 also has a thickness 1034. In the embodiment illustrated in FIG.
• microfilter 1016 has pores 1028 with non-uniform cross-sectional shapes. Each pore 1028 has a first opening in a first surface 1054 of microfilter 1016 and a second opening in a second surface 1056 of microfilter 1016. As shown in FIG. 10D, the width 1048 of pore 1028 at the first surface 1054 is greater than the width 1049 of pore 1028 at the second surface 1056. Microfilter 1016 also has a thickness 1036. In the embodiment illustrated in FIG. 10E, microfilter 1018 has pores 1029 with non-uniform cross-sectional shapes. Each pore 1029 has a first opening 1045 in a first surface 1053 of microfilter 1018 and a second opening 1047 in a second surface 1057 of microfilter 1018. As shown in FIG. 10E, the width 1045 of pore 1029 at the first surface 1053 is smaller than the width 1047 of pore 1029 at the second surface 1057. Microfilter 1018 also has a thickness 1038.
• FIGS. 12A-12K are cross-sectional views illustrating multiple stages in a process for manufacturing a multi-layer microfilter 1270 in accordance with embodiments of the present invention.
• FIG. 12L is a top view of multi-layer microfilter 1270 in accordance with embodiments of the present invention.
• FIG. 11 is a flow chart illustrating a process 1100 for manufacturing a multi-layer microfilter 1420 in accordance with embodiments of the present invention.
• FIGS. 14A and 14B are cross-sectional views of a multi-layer microfilter 1420 in accordance with embodiments of the present invention.
• FIG. 14C is a top view of a multi-layer microfilter 1420 of FIGS. 14A and 14B.
• the exemplary process of FIG. 11 will be described below with reference to FIGS. 12A-12F and FIGS. 14A-14C.
• FIGS. 13A and 13B are top views illustrating multiple stages in the process of FIG. 11.
• a first microfilter 120 is formed on a substrate 180 from a layer of epoxy-based photo-definable dry film.
• first microfilter 120 may be formed on substrate 180 by a process similar to the process 200 of FIG. 2A, described above, omitting the removal of microfilter 120 from substrate 180 at block 280.
• microfilter 120 comprises a polymer layer including a plurality of apertures.
• a mask with a pattern configured for forming a plurality of elongate trenches in the dry film 100 may be used instead of mask 199 with pattern 198 configured for forming a plurality of pores in dry film 100.
• the mask may have a pattern including elongate strips of metal so that corresponding elongate trenches may be formed in dry film 100 when dry film 100 is exposed through the mask.
• FIG. 13A is a top view of microfilter 120 formed at block 1120 in accordance with embodiments of the present invention.
• microfilter 120 includes a plurality of elongate trenches 1222 and is disposed on a substrate 180 exposed through trenches 1222.
• FIG. 12A is a cross-sectional view of microfilter 120 taken along line 12A of FIG. 13A
• FIG. 12B is a cross-sectional view of microfilter 120 taken along line 12B of FIG. 13A. As shown, line 12B is perpendicular to line 12A.
• dry film 1210 (which may be referred to herein as "dry film 1210") is laminated on microfilter 120, as shown in FIG. 12C.
• dry film 1210 is capable of bridging over features formed in the surface on which it is laminated. In such embodiments, dry film 1210 does not significantly fill trenches 1222 when laminated on microfilter 120.
• a second microfilter 1230 is formed from the layer of epoxy-based photo-definable dry film 1210, as described below.
• microfilter 1230 comprises a polymer layer including a plurality of apertures. As shown in FIG.
• dry film 1210 is exposed to energy through a mask 1290 to form an exposed dry film 1212 having a pattern 1218 of polymerized portions and non-polymerized portions 1216, as described above in relation to block 240 of FIG. 2A.
• dry film 1210 is a negative resist.
• dry film 1210 may be a positive resist and a different mask configured for use with a positive resist may be used.
• dry film 1210 is exposed to energy in the form of ultraviolet (UV) light through an optical mask 1290 having a mask portion 1295 that is transparent to UV light and a mask pattern 1293 including a plurality of elongate strips that are opaque to UV light.
• dry film 1210 may be exposed to X-rays through an X-ray mask instead of being exposed to UV light through optical mask 1290.
• FIGS. 12A-12L a polymeric microfilter 1230 having a plurality of trenches 1232 extending through microfilter 1230 is formed from exposed dry film 1212, as described above in relation to block 260 of FIGS. 2 A and 2B, in one variation of block 1160.
• FIG. 13B is a top view of first and second microfilters 120 and 1230 in accordance with embodiments of the present invention.
• microfilter 1230 includes a plurality of elongate trenches 1232 and is disposed on first microfilter 120, which is exposed through trenches 1232.
• FIG. 13B is a top view of first and second microfilters 120 and 1230 in accordance with embodiments of the present invention.
• microfilter 1230 includes a plurality of elongate trenches 1232 and is disposed on first microfilter 120, which is exposed through trenches 1232.
• FIG. 12E is a cross- sectional view of microfilters 120 and 1230 taken along line 12E of FIG. 13B
• FIG. 12F is a cross- sectional view of microfilters 120 and 1230 taken along line 12F of FIG. 13B. As shown, line 12F is perpendicular to line 12E.
• the thickness of each layer can be different.
• substrate 180 may be removed from microfilter 120, as described above in relation to block 280 of FIG. 2A, to form a multi-layer microfilter 1420 shown in FIGS. 14A-14C.
• multi-layer microfilter 1420 includes second microfilter 1230 disposed on first microfilter 120.
• multi-layer microfilter 1420 includes apertures 1240 extending through multi-layer microfilter 1420 where trenches 1222 and 1232 intersect.
• microfilter 1240 comprises a polymer layer including a plurality of apertures.
• FIG. 14A is a cross-sectional view of microfilter 1420 taken along line 14A of FIG. 14C
• FIG. 14B is a cross-sectional view of microfilter 1420 taken along line 14B of FIG. 14C. As shown, line 14B is perpendicular to line 14A. The thickness of each layer can be different.
• a microfilter 1270 having a non-linear passage 1280 may be formed, as illustrated in FIGS. 12G- 12L.
• multi-layer microfilter 1270 shown in FIG. 12L, is formed by forming a third microfilter 1240 on first and second microfilters 120 and 1230 and removing substrate 180.
• a layer of epoxy-based photo-definable dry film 1215 is laminated on second microfilter 1230, as shown in FIG. 12G.
• third microfilter 1240 is formed from dry film 1215, as described above in relation to the formation of second microfilter 1230 and the processes of blocks 240 and 260 of FIGS. 2A and 2B.
• third microfilter 1240 includes a plurality of elongate trenches 1242 that are substantially perpendicular to trenches 1232 and substantially parallel with trenches 1222. Additionally, in certain embodiments, trenches 1242 are offset from trenches 1222 such that trenches 1242 are not directly above trenches 1222, as shown in FIG. 12H.
• substrate 180 may be removed from microfilter 120, as described above in relation to block 280 of FIG. 2A, to form multi-layer microfilter 1270.
• FIG. 12L is a top view of multi-layer microfilter 1270.
• FIG. 12J is a cross-sectional view of multi-layer microfilter 1270 taken along line 12J of FIG. 12L, and
• FIG. 12K is a cross-sectional view of multi-layer microfilter 1270 taken along line 12K of FIG. 12L. As shown, line 12K is perpendicular to line 12J.
• multi-layer microfilter 1270 includes non-linear passages 1280 extending through each of microfilters 1240, 1230, and 120 so as to extend from a first surface 1272 to a second surface 1274 (see FIG. 12J) of multi-layer microfilter 1270.
• each non-linear passage 1280 is defined by a first aperture 1282 at an intersection of trenches 1242 and 1232, a second aperture 1284 at an intersection of trenches 1232 and 1222, and a portion of trench 1232 connecting the first and second apertures.
• each non-linear aperture 1280 is interconnected with many other non-linear passages 1280 via trenches 1232.
• the respective thicknesses of microfilters 120, 1230 and 1240 can be the same or different, the trenches of a microfilter may or may not all have the same size and/or shape, the trenches of different microfilters of the multi-layer microfilter may or may not all have the same size and/or shape.
• elongate trenches 1242 may have a width of 5-7 microns and a length greater than 7 microns, wherein the length and the width are both perpendicular to the thickness of the microfilter.
• the trenches of thicknesses of microfilters may be non-linear, and the trenches of adjacent microfilters may be oriented at an angle other than 90 degrees with respect to one another.
• one or more of microfilters 120, 1230 and 1240 may include pores like any of the pores illustrated in FIGS. 9A-9D instead of trenches, and, in some embodiments, multi-layer microfilter 1270 may include more than three microfilters disposed on one another.
• each of microfilters 120, 1230 and 1240 may be formed from the same type of epoxy-based photo- definable dry film.
• FIG. 15 is a cross-sectional view of a microfiltration structure including a microfilter and a support structure in accordance with embodiments of the present invention.
• microfiltration structure 1510 includes a microfilter 1520 having pores 1522 and disposed on a support structure 1530 configured to provide structural strength to microfilter 1520.
• support structure 1530 may be integrated with microfilter 1520.
• support structure 1530 is a grid support structure.
• microfiltration structure 1510 may be formed by a process similar to the process described above in relation to FIGS. 12A-12F and FIGS. 14A-14C.
• microfilter 1520 and support structure 1530 are each formed from a layer of epoxy-based photo-definable dry film and patterned using an appropriate mask.
• microfilter 1520 is formed on support structure 1530 or support structure 1530 is formed on microfilter 1520.
• the microfilter can be a combination of pores with other structure elements above or below the layer that form the pores to form many filtration devices.
• a few examples of two layers or three layers structures with and without pores are illustrated in FIGS. 16A-16J. These devices have applications for isolating cells from body fluids and for biological assays.
• FIG. 16A is an exemplary embodiments of a two layered microfilter, showing a side view of microfilter 2561 containing posts 2575 on microfilter base 2572 with pores 2573.
• FIG. 16B shows the top view of posts 2575 on microfilter base 2572 with pores 2573.
• the pore can be circular, square, rectangle, etc.
• the posts can also of a variety of shapes such as circles, squares, rectangles, etc.
• the arrangement of the posts and the density can be regular, varied spatially or random as long the posters are not closer than 30 ⁇ and the posts don't cover the pores.
• the pores can have various shapes, sizes and distributions.
• the posts can also have various shapes, sizes and distributions.
• FIG. 16C is another exemplary embodiments of a two layered microfilter, showing the top view of a microfilter 2563 where pore is rectangular shaped 2573, but not distributed everywhere on the base support 2572, and posts 2575 is also not distributed everywhere.
• the pores can have various shapes, sizes and distributions.
• the posts can also have various shapes, sizes and distributions.
• FIG. 161 is an exemplary embodiments of a two layered device 2580, showing the side view of bottom layer 2581 without pores.
• the top layer 2582 forms wells 2583.
• FIG. 16J shows the top view.
• This well structure can be fabricated using the same procedure described in FIGS. !A-ID followed by procedure described in FIGS. 12A-12F except no mask is needed for FIG. IB.
• Pore dimensions depends on the needs of specific applications. For example, pore diameter of 7-8 microns is the commonly preferred for circulating tumor cells from human blood, while circulating tumor cells originated in mice are smaller than in humans, so smaller pores are preferred for mice studies. For rectangular pores, width of 5-7 ⁇ are preferred for collecting circulating tumor cells from human blood and the length is not critical as long as the filters don't deform during filtration.
• FIGS. 16D-16F is another exemplary embodiment of a two layered microfilter with different patterns in each layer of the material according to an exemplary implementation.
• FIG. 16D showing the first side view 2501 of a two layer microfilter with the top layer 2551 and bottom layer 2552. The top layer are of strips with slot opening 2555.
• FIG. 16E is the second side view 2502 of the two layer microfilter turned 90 degrees. The bottom layer 2552 has open slots 2553.
• FIG. 16F shows the top view of the microfilter 2500. The cross strips forms effective rectangular pores 2554.
• 2555 between two top strips of 2551 are 5-7 ⁇ for isolation of tumor cells.
• Small width of 10- 20 ⁇ for 2551 would allow high filtration rate, but a wide range of with for 2551 are functional.
• the width of 2552 can vary, but for high filtration rate, the width may be 5-20 ⁇ .
• the gap between two strips of 2552 that form the pore 2553 can also vary. For high filtration rate, the gap can be from 10-60 ⁇ .
• the thickness of 2551 about 10 ⁇ may be preferable. In an exemplary implementation, thickness of bottom layer 2552 can advantageously be approximately the same as the gap between two strips of 2552.
• FIGS. 16G-16H are side view and top view of an exemplary device 2540 consisting of microfilter 1541 with pores 2542 at the bottom of wells 2544.
• the structure shown in FIG 16H is repeated over the whole microfilter area.
• An application of the device 2540 is to filter the cells and followed by culture of the cells in the wells 2544. Again the pores can have a variety of shapes and sizes.
• the size, shape and depth of the well 2544 can also be varied as appropriate for different uses.
• the density of the wells 2583 can also be varied.
• An exemplary application of devices 2500, 2540, 2561 and 2563 includes rare cell isolation where the mechanism of isolation of the cells is based on size.
• FIGS. 16T16J are side view and top view of an exemplary device 1580 consisting of wells 2583 formed by 2582 with solid bottom 2581. These can be used for a variety of biological assays. The size, shape and depth of the wells 2583 can be varied. The density of the wells 2583 can also be varied.
• FIGS. 16A-16J Various materials for forming devices described in FIGS. 16A-16J above can include epoxy-based photo-definable dry films and other types of photo-definable dry films. The methods to fabricate those structures using photo-definable dry films are described above and in, for example, PCT/US 11/20966.
• surface functionalization of a polymeric microfilter may provide a surface of the microfilter with surface properties desired for a particular application of the microfilter. Some materials can be directly disposed on the microfilter surfaces. Other times, the microfilter surfaces need to be treated.
• the surface of the polymeric microfilter can be functionalized by performing a plasma treatment on the surface of the microfilter to activate the surface to enable chemical compounds and/or organic materials to attach to the surface.
• another surface modification technique is to coat the microfilter with a thin layer of a metallic substance.
• FIGS. 17 and 18 are cross-sectional views of coated microfilters in accordance with embodiments of the present invention.
• microfilter 1655 includes a coating 1600 on surface of one layer microfilter 1655.
• Coating 1600 can also be disposed on multi-layered microfilters.
• Coating 1600 can also be disposed on microfilters produced by other methods and by other materials.
• the multi-layer device is formed from epoxy-based photo-definable dry film, as described above in relation to FIGS. 16A-16J.
• microfilter 1672 includes a coating 1600 on surface of the top structure and flat portions of the microfilter 1672.
• Coating 1600 can also be disposed on microfilter structures produced by other methods and by other materials. Coating 1600 can also be disposed on the surface of structure shown in FIGS. 16I-16J even without pores.
• coating 1600 may be formed from a metallic substance, a nanoparticle colloidal substance, chemical compound, or an organic substance.
• these surface coatings can be used to attach analyte recognition elements, DNA, aptamers, surface blocking reagents, etc.
• coating 1600 may include analyte recognition elements, DNA, aptamers, surface blocking reagents, etc.
• the surface coatings may be used to attach, for example, macromolecules such as polypeptides, nucleic acids, carbohydrates and lipids.
• nucleic acids that may be used as analyte recognition elements include, for example, DNA, cDNA, or RNA of any length that allows sufficient binding specificity. In such embodiments, both polynucleotides and oligonucleotides can be used as analyte recognition elements.
• analyte recognition coatings or elements may include, for example, biological particles such as a cell, a cell fragment, a virus, a bacteriophage or tissue.
• analyte recognition coatings or elements may include, BSA, fetal bovin serum (FBS), selectins including P-selectins, E-selectins, L-selectins, nanoparticles, nanotubes, halloysites, dendrimers, chemical linkers or other chemical moieties that can be attached to a microfilter and which exhibit selective binding activity toward a target analyte.
• BSA fetal bovin serum
• selectins including P-selectins, E-selectins, L-selectins, nanoparticles, nanotubes, halloysites, dendrimers, chemical linkers or other chemical moieties that can be attached to a microfilter and which exhibit selective binding activity toward a target analyte.
• coating 1600 may be formed from a metallic substance including gold, nickel, etc.
• coating 1600 includes gold coated on chromium.
• coating 1600 may be formed from carbon nanotubes.
• coating 1600 is disposed on one surface of microfilters.
• one or more surfaces of microfilters may be coated with coating 1600.
• microfilters may be completely coated with coating 1600.
• Coating 1600 may be disposed on one or more surfaces of any of the microfilters described herein in accordance with embodiments of the present invention, including multi-layer microfilters.
• coating 1600 may be disposed on one or more surfaces of multi-layer microfilter 1620.
• examples of chemical compounds and organic materials that may be useful for assays when deposited on the surface of a microfilter include are self- assembled monolayers with a range of functionality including amine, carboxyl, hydroxyl, epoxy, aldehyde, and polyethylene glycol (PEG) groups. These compounds and materials may be deposited on the surface of a microfilter using silane chemistry with solution immersion or vapor deposition. In certain embodiments, for example, grafting PEG-triethoxysilane onto an oxidized polymer renders the surfaces hydrophilic in a controlled manner. In other embodiments, a surface of a polymeric microfilter can be functionalized with avidin, biotin, protein A, protein G, antibodies, etc.
• coating a surface of a microfilter with a metallic substance may provide other benefits in addition to facilitating the attachment of chemical compounds and/or organic materials.
• a layer of a metal metallic substance having an appropriate thickness, can block transmission of light through the microfilter.
• a thickness sufficient to block the transmission of light is about 40 nm. In other embodiments, this thickness may vary depending on the substance used.
• metallic substances are generally electrically conductive. In some embodiments, when the metallic substance is electrically conductive, the coating may reduce or eliminate charging of the surface of the microfilter.
• a microfilter may be coated with a thin layer of PARYLENE.
• a microfilter may be coated with a thin layer of fluorinated ethylene propylene (FEP), polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), or another similar material.
• FEP fluorinated ethylene propylene
• PTFE polytetrafluoroethylene
• PFA perfluoroalkoxy
• single layer or multi-layered microfiltration devices can be coated with an antibody against surface markers on the CTCs to further improve the collection of live CTCs.
• capture efficiency of live CTCs may be improved.
• useful surface markers may include antibodies against EpCAM, HER2, EGFR, KRAS, Vimentin, and MUC-1, but not limited to these surface markers, P-selectin, E-selectin, other selectins, ligends, aptamers, etc.
• the microfiltration can capture CTCs through size exclusion and surface markers simultaneously.
• the steps of filtration of large rare cells from body fluids on the microfilter or concentrated in a reduce volume with reduced contaminants can be performed with minimal human intervention if the microfilter can be installed in a microfiltration device.
• the present invention details a method to use microfilters using filter holders.
• FIG. 19 can be used in a variety of ways to analyze rare cells such as CTCs from body fluids.
• Rare cells can be collected on the epoxy-based photo-definable dry film microfilters and other precision microfilters. Following the rare cells collection, rare cells can be analyzed using the following methods:
• Immunofluorescence staining to look for expression of biomarkers to identify cancer subtype, or to determine biomarker mutation, or cell type. These information can be used to determine cancer therapy and monitor treatment; immunofluorescent staining can be used to perform enumeration of CTCs, based on DAPI (positive), Cytokeratins (CKs) (positive), such as CK8, 18, 19 and others, and CD45 (negative) cells, and count the number per ml of blood to monitor cancer treatment and recurrence;
• DAPI positive
• Cytokeratins CKs
• CD45 negative
• FISH Fluorescent in situ hybridization
• mRNA FISH can also be performed to determine the marker is over-expressing.
• Nucleic acid assays for mRNA, microRNA and genomic DNA biomarkers, and gene mutations to identify cancer subtypes to determine therapy and monitor treatment;
• Culture rare cells such as CTCs, to increase the number of cells. These cells can then be used to perform the assays described in bullets above. In addition, viable cells can be used to determine the effect of drugs on the cancer cells to determine therapy.
• rare cells collected on epoxy-based photo-definable dry films can be performed sequentially to obtain different information from the same cells.
• Some examples are: (1) Perform enumeration and for a cancer surface biomarker, followed by FISH assay and finally histopathological staining, (2) perform immunofluorescent staining to count the number of rare cells and at the same time determine over-expression of biomarker(s), mutated biomarker(s), cell type, and/or other information, followed by histopathological staining, (3) perform FISH to identify a marker followed by histopathological staining.
• filtration devices to hold the filter and perform filtration are designed to have the following features:
• This filter holder has a large opening in the inlet unit. This is to allow easy access of the reagents to microfilter surface and visual access to the microfilter, where the cells are collected, and allow visualization of the filter from above.
• the filter holder will allow performance of at least some types of assay in the filter holder. • The filter holder will allow different attachment of input sample holder above the filter holder, and syringe, stopper on the filter flask connected to a vacuum pump or vacutainer holder below.
• a filter holder allows for (i) filtration of the liquid sample through the microfilter, (ii) ability to perform assays with the microfilter in the holder, (ii) 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, and (vi) filter holder material capable of tolerating temperature required for the assays, and (vii) filter holder material capable of tolerating chemicals used in the assays.
• FIGS. 20A-20C show three components that forms a filter holder according to an exemplary embodiment of the present invention, including: an inlet unit 3010, a nut 3020 and an outlet unit 3030 (top view FIG. 20C and the bottom view FIG. 20D).
• a syringe or vacutainer holder with a Lure-Lock can be attached to the outlet unit of the filter holder.
• an output unit can include a cut out on the top ring, for example, to allow access to the microfilter to allow easy removal of microfilters from the filter holder.
• FIGS. 21A-21E show the assembly of the filter holder according to an exemplary embodiment of the present invention where a gasket 3110 is placed inside the outlet unit as shown in FIG. 21 A.
• a round microfilter 3120 with the appropriate diameter for the filter holder is placed above the gasket, FIG. 21B.
• a second gasket 2130 is place above the microfilter 3120, FIG. 21C.
• the inlet unit 3010 is placed into the outlet unit 3030 by properly aligning the two components as shown in FIG. 21D.
• the nut 3020 is installed to tighten the filter holder system to prevent leakage of the liquid sample around the microfilter inside the holder. While one gasket may be sufficient, a plurality of gaskets may be used, for example to prevent leakage, as needed.
• the gasket can be of different material or design. In an exemplary filter holder design, a microfilter installed in a filter holder system will remain flat, experience compression to form a tight seal, but not experience any twisting force.
• the filter holder can have different sizes to accommodate different microfilter sizes.
• the common microfilter sizes are 0.5 inch (13 mm) and 1 inch diameters, but are not limited to these dimensions.
• the filter outlet unit can have more than one opening.
• the opening is to allow easy removal of microfilter from filter holder and prevent the microfilter from becoming twisted by fixing in place the inserted inlet unit.
• the shape and dimensions of different component of the filter holder can vary.
• One of the features of the filter holder concept according to exemplary embodiments of the present invention is pressing down on the inlet unit to prevent leakage.
• a nut is used to hold down the inlet unit.
• the inlet unit can also be held down by changing the nut to a snap-on part.
• the outlet 3030 can have a female connector or a male connector.
• the filter holder can be designed to accommodate one gasket under the microfilter or two gaskets, one above and one below the microfilter.
• a filter support structure can be added to the inside of output units of the filter holders. Support structure may not be needed for epoxy-based photo-definable dry film microfilters because the material is sufficiently strong.
• the inlet unit 3010 can be combined with inlet adaptor 3050, in FIG. 23, into one piece.
• Exemplary embodiments of filter holders described in this application are applicable to most filters and microfilters. Certain exemplary embodiments can be particularly suitable to very thin and strong microfilters such as epoxy-based photo-definable dry film microfilters.
• FIGS. 22A-22B show examples of sample input containers for holding the liquid sample and wash buffers.
• FIG. 22A shows an inlet container 3200 with the shape similar to a syringe with sample inlet opening 3210 and connection to the filter holder 3220.
• FIG. 22B shows an inlet container 3300 with wider opening 3310 and connection to the filter holder 3320 to allow easy access to the microfilter.
• FIGS. 22A-22B show examples of sample input container made as one piece.
• FIG. 23 shows an example of a sample input container 3420 that can also be constructed out of an off the shelf syringe 3410 without a plunger and an inlet adaptor 3050 with a male Lure-Lock.
• the inlet adaptor can have various designs as long as it does not leak and it can be easily removed from the filter holder.
• the shape at the bottom of the sample input containers 3200, 3300, and the inlet adaptor 3050 should have a shape to facilitate tight fit into the inlet unit 3010 on the top of the assembled filter holder 3100 in FIG. 21E.
• an O-ring can be provided to facilitate the tight fit into the inlet unit 3010.
• the filtration system 3500 can assume the configuration shown in FIG. 24.
• the input container 3420 constructed as shown in FIG. 23, is connect to the top of the assembled filter holder 3100 from FIG. 21E, and the bottom of the assembled filter holder 3100 shown in FIG. 21E is connected to a waste syringe 3510 with plunger 3520..
• FIG. 25 shows a filtration system with just the filter holder 3100 and the waste syringe 3510.
• the opening 3140 allows performance of many assay steps directly on the microfilter.
• the filtration can be performed manually by drawing the plunger, but manual operation may not provide consistent speed.
• the filtration system 3600 using syringe pump 3610 in FIG. 26 can provide more consistent speed for pulling the plunger 3520 by a pusher block 3620.
• the syringe pump can have just infusion or both infusion and withdrawn functions.
• FIG. 27A shows another exemplary method for drawing blood through the microfilter by connecting a vacutainer holder 3710 to the outlet of the filter holder 3730.
• a vacutainer 3740 is inserted into the vacutainer holder 3710, shown in FIG. 27B, the vacuum in the vacutainer will draw the liquid sample into the vacutainer and the microfilter will collect the rare cells.
• FIG. 28A shows a filtration device 3800 that can perform filtration at the time of getting blood draw.
• the filtration device combines a filter holder 3830 with the inlet adaptor 3820 and vacutainer holder 3810.
• a needle 3850 with female Luer-Loc can be attached to the inlet adaptor 3820.
• FIG. 28B shows a vacutainer 3840 inserted into the vacutainer holder 3810.
• FIG. 29A illustrates an example of a method to obtain samples and shipping to clinical laboratories for testing including : (i) collecting body fluid, (ii) sending sample to testing laboratory, (iii) filtering rare cells out of body fluids, and (iv) performing an assay.
• FIG. 29B describes an alternative method for obtaining samples, and for testing in clinical laboratories including: (i) collecting body fluid, (ii) filtering the rare cells from the body fluid at the collection site, (iii) sending filter holder with microfilter or just the microfilter with captured rare cells to testing laboratory, and (iv) performing the assay in the testing laboratory.
• the filter holder with microfilter or just eh microfilter with captured rare cells can be sent to testing laboratory. Assays will be performed in the testing laboratory.
• microfilters can be formed from epoxy-based photo-definable dry film having a thickness between 1-500 ⁇ .
• such microfilters can be formed using UV light to expose the dry film (i.e., using UV lithography).
• X-rays i.e., X-ray lithography
• relatively thick microfilters may provide more structural strength than thinner microfilters, but may also utilize higher pressure during filtration.
• epoxy-based photo- definable dry films are a preferred material from which to form microfilters in accordance with embodiments of the present invention.
• properties of epoxy-based photo- definable dry film that make it a suitable material from which to form microfilters for medical diagnostic applications are that it is photo-definable by UV light, it is clear, it has a high tensile strength of 75 Mpa, it can be laminated to itself, it can be directly coated on a substrate, and it has no auto-fluorescence in the visible wavelengths.
• the processes described above in accordance with embodiments of the present invention may be used to form microfilters, the processes described above may also be used to manufacture other kinds of freestanding patterned polymeric films.
• Microfilters formed in accordance with exemplary embodiments of the present invention have many possible applications.
• exemplary applications for such microfilters include medical applications, water filtration applications, beer and wine filtration applications, pathogen detection applications, etc.
• FIG. 30A is a flowchart illustrating a filtration process 1700 using a microfilter in accordance with exemplary embodiments of the present invention.
• a liquid may be passed through a microfilter formed from a layer of epoxy-based photo- definable dry film, in accordance with any one of the embodiments described above, having a plurality of apertures.
• the liquid may be pushed through the microfilter.
• the liquid may be drawn through the microfilter. The draw can be produced by a syringe or by vacuum.
• the liquid may be passed back and forth through the microfilter one or more times.
• the particulates retained on the microfilters may be backwashed using appropriate liquid.
• the process illustrated in FIG. 30A may be used to perform an assay using the microfilter.
• the process may be used to filter cells, such as CTCs, from a solution including a patient's bodily fluid. This can be accomplished using vacutainer as shown in FIG. 28A-28B, or by placing the filter on a support above a vacuum pump.
• FIG. 30B is a flowchart illustrating a filtration process 1800 using a microfilter in accordance with exemplary embodiments of the present invention.
• a microfilter formed from a layer of epoxy-based photo-definable dry film in accordance with any one of the embodiments described above, is positioned in a filter holder.
• the filter holder includes an inlet, an outlet, and securely holds the microfilter around the edges of the filter.
• a liquid may be input into the filter holder through the inlet.
• the liquid is passed through the microfilter.
• the liquid is a bodily fluid or a solution including a bodily fluid.
• the liquid is drawn through the microfilter by applying negative pressure at the outlet of the filter holder such that all or substantially all of the liquid is drawn through the pores of the microfilter. In other embodiments, the liquid is pushed through the microfilter.
• the microfilter may also be removed from the filter holder. For microscope imaging, the microfilter may be placed on a glass slide.
• FIG. 30C is a flowchart 1900 illustrating examples for performing some types of assays in the filter holder 1910. After the assay, the filter can be removed from the filter holder 1920 for analysis.
• Enzymatic activity assay, histopathology staining (colorimatric staining), and immunofluorescent staining for applications such as determination of biomarker expression, EPISPOT and enumeration, can follow the flow chart steps in FIG. 30C after step shown in FIG. 30B.
• These assays can be performed in a filter holder as described. These assays can also be performed on glass slide, or plate after removing the microfilter from the filter holder follow the flow chart shown in FIGS. 30A or 30B.
• Nucleic acid assays, sequencing, fluorescence in situ hybridization (FISH), mRNA in situ hybridization, and culture require removing the microfilter with captured rare cells from the filter holder and perform the assays in each of their own appropriate ways as shown in flow chart of FIGS. 30A or 30B.
• the protocol to isolate cells is as follows using the syringe pump: 1. Assemble the microfilter in the filter holder as shown in FIGS. 21A-21E;
• the syringe pump can be operated manually or can be automated.
• the filtration can be performed manually.
• skip step 2 To perform the assay manually, skip step 2.
• complete assembled system looks like FIG. 24.
• the filter holder 3100 can also be placed on a stopper on a filter flask connect to a vacuum pump with the inlet container 3420.
• the liquid sample and the wash buffer can be placed into the inlet container 3420.
• the vacuum pump turned on to draw the liquid through the filter. If the inlet container is not used, the liquid sample and the wash buffer can be pipette into the reaction well 3010 while the vacuum is on.
• the filtration can be performed using the vacutainer systems 3700 as shown in
• FIG. 27A The steps are:
• vacutainer 3740 Insert vacutainer 3740 into the vacutainer holder 3710, as shown in FIG. 27B.
• the filtration can be performed at the time of blood draw using the vacutainer systems 3800 as shown in FIG. 28A.
• the steps are:
• vacutainer 3840 into the vacutainer holder 3810, as shown in FIG. 28B.
• FIG. 30C shows that many assay steps can be performed in the filter holder after the steps in the flow chart of FIGS. 30A-30B. The details of the assay steps can vary depending on the assay. This is performed using the configuration shown in FIG. 25. The steps will vary depending on the assay. The general steps are described below.
• Incubation Place reagents into the reservoir 3140 above the filter and incubate. The incubation of with reagents above the microfilter in the filter holder is possible because the apoxy-based photo-definable dry film microfilter is hydrophobic. Reagents will not leak through the microfilter.
• wash buffer For small amount of wash buffers after the incubation, wash buffer can be placed directly into the reservoir 3140 and sucked out by negative pressure. This can be repeated. When larger volumes of washing buffers are required, place the input container back on the filter holder to form the system shown in FIG. 24 and perform washing step by sucking out the wash buffer placed into the input container.
• Nucleic acid assays and sequencing are suited for performing the assay from the lysed cells.
• the conceptual protocol follows the steps of collecting the cells on the microfilter. After taking the microfilter out of the filter holder, place the microfilter containing rare cells i6 an Appendorf centrifugation tube with lysis buffer. The rest of the steps for the nucleic acid assays are the same as common samples.
• the conceptual protocol follows the steps of collecting the cells on the microfilter. After taking the microfilter out of the filter holder, place the microfilter containing rare cells in culture medium. Cells can also be backwashed out of the filter into culture medium. In some situations the rare cells need to be removed from the microfilter and inject into an animal such as mice.
• the particulates retained on the microfilter may then be subject to processing and or analysis to analyze any cells or other materials, substances, etc. collected by the microfilter.
• the analysis may be performed in the filter holder device or microfiltration chip.
• the analysis may also be performed outside the filter holder after the microfilter is removed from the filter holder. Exemplary applications of this process in accordance with embodiments of the present invention will be described below.
• a microfilter formed from a layer of epoxy-based photo-definable dry film in accordance with any one of the embodiments described above may be used for medical diagnostics and/or prognostics.
• the microfilter may be used to collect certain types of cells from bodily fluids based on cell size.
• the microfilter can be used for isolating and detecting rare cells from a biological sample containing other types of cells.
• the microfilter can be used to filter a fluid sample, and the collected cells can be used in a downstream processes such as cell identification, enumeration (cell counting), characterization of the collected cells, culturing the collected cells, separating the cells into individual cells or groups of cells, or use the of cells in other ways.
• a downstream processes such as cell identification, enumeration (cell counting), characterization of the collected cells, culturing the collected cells, separating the cells into individual cells or groups of cells, or use the of cells in other ways.
• the final enriched target cells can be subjected to a variety of characterization and manipulations, such as staining, immunofluorescence of markers, cell counting, DNA, mRNA, microRNA analysis, fluorescence in-situ hybridization (FISH), immunohistochemistry, flow cytometry, immunocytochemistry, image analysis, enzymatic assays, gene expression profiling analysis, sequencing, efficacy tests of therapeutics, culturing of enriched cells, and therapeutic use of enriched rare cells.
• depleted plasma protein and white blood cells can be optionally recovered and subjected to other analysis, such as inflammation studies, gene expression profiling, etc.
• the microfilter can be held in a filter holder for medical diagnostics and/or prognostics.
• the filter holder may include a built in support for the microfilter.
• the filter holder may have gasket above and below the filter.
• the microfilter may be used to collect circulating tumor cells (CTCs) in blood.
• CTCs circulating tumor cells
• a blood sample typically in the range of 1-10 ml, is taken from a patient.
• the blood sample is then drawn through the microfilter by applying negative pressure, such as a sucking force.
• the blood is pulled through the microfilter via an outlet.
• passing the blood through the filter by pushing can cause cell rupture except at very low pressure or low speed.
• the microfilter may include pores 7-8 ⁇ in diameter for enriching circulating tumor cells and fetal cells; however, the microfilter pore size and shape can be varied for these applications as well.
• CTCs collected by the microfilter can be enumerated on the microfilter.
• the microfilter used to demonstrate filtration efficiency was a microfilter having a 7-8 micron diameter pores separated by 20 microns and arranged within a 9 mm diameter area.
• the microfilter was placed into a filter holder.
• a prestained MCF-7 cell line was spiked into 7.5 ml of whole blood.
• the blood was diluted 1: 1 with a buffer solution.
• One exemplary buffer solution is phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• Another exemplary buffer solution is a mild fixation buffer to make the CTCs slightly more rigid.
• the liquid sample was drawn through the microfilter using negative pressure at approximately 10 ml/min. Afterwards, the filter was washed twice in a buffer solution. The microfilter was removed from the holder and mounted onto a microscope slide to be counted. The recovery rate of live MCF-7 cells was 85 +3 . If the blood is mildly fixed, the capture efficiency of MCT-7 cells increases to 98 +2 .
• collected CTCs can be subjected to a variety of analyses and manipulations, such as immunofluorescence, genetic characterization and molecular phenotyping, fluorescence in-situ hybridization (FISH), mRNA FISH, in-situ hybridization (ISH), mRNA ISH, immunohistochemistry (IHC), flow cytometry, immunocytochemistry, colorimetric staining, histopathological staining (for example, hematoxylin and eosin staining), image analysis, epithelial immunospot (EPISPOT), enzymatic assays, gene expression profiling analysis, efficacy tests of therapeutics, culturing of enriched cells, and therapeutic use of enriched rare cells.
• depleted plasma protein and white blood cells can be optionally recovered, and subjected to other analysis such as inflammation studies, gene expression profiling, etc.
• CTCs collected from blood can be stained to identify them as potential tumor cells and not blood cells. It is possible to identify the CTCs by morphology using colorimetric staining. Other methods are based on fluorescence staining. Some typical fluorescence staining methods to identify the cells as tumor cells use DAPI to identify the nucleus and us cytokeratins 8, 18 and 19 conjugated to a fluorescent dye to identify them as epithelial cell. Since normal epithelial cells are not fund in blood, epithelial cells found in blood are accepted as tumor cells. CD45 antibody is used to identify white blood cells, eliminating the blood cells retained on the microfilter as CTCs.
• a cell from blood that is DAPI positive, CK 8, 18, and 19 positive and CD45 negative to be a CTC.
• Other markers such as EpCAM, MUC-1, and others can be included to further provide specificity and increase of fluorescence signal.
• captured CTCs are stained to specifically identify the origin of the tumor cells, such as breast, prostate, colon, etc.
• PSA marker on the cell would identify its origin as prostate.
• specific markers can be found either on the surface or inside the cells.
• captured CTCs can be characterized to determine if it contains specific mutations of the DNA. This can be identified by for DNA, mRNA, and microRNA expression by PCR, by sequencing, or by antibodies, ligends, aptamers and others that recognize the mutated proteins.
• captured CTCs can be characterized to determine over- expression of genes. Sometimes the number of copies of a gene is more than it should be for each cell. When there are more copies of the gene, it produces more copies of mRNA, which in turn produces more copies of the protein. The number of copies of the gene can be determined by FISH or by ISH, The amount of mRNA produced can be obtained by PCR, mRNA FISH and mRNA ISH. The amount of proteins produced can be determined by immunofluorescent stains for that protein. Cells over-expression a marker will stain brighter for that marker than normal tissue. One example of over expression is HER-2 in some subtypes of breast cancer. [00185] In certain embodiments, captured CTCs can be characterized to determine if they are viable cells.
• captured CTCs can be determined if they are viable.
• Viability can be determined by trypan blue staining, or culture.
• captured CTCs can be determined if they are stem cells. They can be stained for stem cell marker phenotype (CD44 + /CD24 "/low or CK19 + /MUC- ⁇ )
• viable CTCs may be captured by microfilter coated by analyte recognition elements, such as antibodies, ligends, aptamers, etc..
• the analytes of interest are to be secreted by the CTC.
• a secondary analyte recognition element can be used to produce a detectable signal if the analyte is produced by the CTC.
• the signal may be produced by fluorescent dyes. The concept is similar to EPISPOT.
• captured live CTCs can be cultured directly on the microfilter to increase the number of CTCs and to evaluate the characteristics of CTCs.
• the CTCs can be backwashed from the microfilter prior to culturing or sorting.
• a microfilter formed from a layer of epoxy-based photo- definable dry film in accordance with embodiments of the present invention may be used in therapeutic applications in which circulating tumor cells are removed from the blood of cancer patients. Circulating tumor cells are the cause of cancer spreading from the original site to other locations such as brain, lung and liver. Most carcinoma cancer patients die from the metastatic cancer.
• microfiltration using a microfilter formed in accordance with embodiments of the invention is a suitable method for removing circulating tumor cells from the blood stream of a patient because the filtration speed is fast and microfilters retain very little white blood cells and almost no red blood cells when used to filter blood.
• Microfiltration for circulating tumor cells in blood can provide a large array of diagnostic, prognostic and research applications.
• previous research reports utilized track etch filters with random pore locations with some overlapping pores and not straight pores, and microfilters with orderly arranged pores produced by reactive ion etching.
• microfilters formed from epoxy- based photo-definable dry film and having precisely arranged pores are used to collect circulating tumor cells in blood.
• One exemplary application of a microfilter formed in accordance with embodiments of the present invention is to monitor effectiveness of treatment by counting the number of CTCs collected in the blood. Large number of CTC per ml of blood can indicate short lifespan. The change of CTCs per ml of blood can indicate whether treatment is working or not. If the number of CTCs is decreasing, it indicates the treatment is having an effect. In contrast if the number of CTCs is increasing, it indicates the treatment is ineffective.
• microfilter formed in accordance with embodiments of the present invention is using the microfilter for capturing cells and subsequently culturing the cells in the filter holder, or culturing the cells after back flushing the cells off the microfilter.
• Various drugs can be applied to the cultured CTCs can be used to evaluate drug efficacy to determine the best treatment for the patients.
• microfilter formed in accordance with embodiments of the present invention is to determine gene mutation in CTCs to determine the appropriate drug.
• One exemplary application of a microfilter formed in accordance with embodiments of the present invention is to determine over expression of a marker when there is drug to treat tumors with the over expression
• One exemplary application of a microfilter formed in accordance with embodiments of the present invention is to determine if the cancer is returning after remission. If the number of CTCs become more than five from 7.5 ml of blood and the CTC count is increasing in time, then the cancer is returning.
• Another exemplary application of a microfilter manufactured in accordance with embodiments of the present invention is capturing circulating endothelial cells. Endothelial cells in the peripheral blood provides information about various medical conditions.
• fetal cells may include primitive fetal nucleated red blood cells.
• Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are 14-60 ⁇ in diameter, larger than peripheral blood leukocytes. Enrichment of circulating fetal cells followed by genetic diagnostic can be used for noninvasive prenatal diagnosis of genetic disorders using PCR analysis of a DNA target or fluorescence in situ hybridization (FISH) analysis of genes.
• FISH fluorescence in situ hybridization
• Another exemplary application of a microfilter manufactured in accordance with embodiments of the present invention is collecting or enriching stromal cells, mesenchymal cells, endothelial cells, epithelial cells, stem cells, non-hematopoietic cells, etc. from a blood sample, collecting tumor or pathogenic cells in urine, and collecting tumor cells in spinal and cerebral fluids.
• Another exemplary application is using the microfilter to collect tumor cells in spinal fluids.
• Another exemplary application is using the microfilter to capture analytes bound to latex beads or antigen caused particle agglutination whereby the analyte coated bead or agglutinated clusters are captured on the membrane surface.
• Red blood cells are highly flexible cells that will readily change their shape to pass through pores. In some diseases, such as sickle cell anemia, diabetes, sepsis, and some cardiovascular conditions, the cells become rigid and can no longer pass through small pores. Healthy red cells are typically 7.5 ⁇ and will easily pass through a 3 ⁇ pore membrane, whereas a cell with one of these disease states will not.
• a microfilter having 5 ⁇ apertures is used as a screening barrier. A blood sample is applied and the membrane is placed under a constant vacuum. The filtration rate of the cells is then measured, and a decreased rate of filtration suggests decreased deformability.
• a microfilter formed in accordance with embodiments of the present invention is leukocyte/Red blood cell separation.
• Blood cell populations enriched for leukocytes are often desired for use in research or therapy.
• Typical sources of leukocytes include whole peripheral blood, leukopheresis or apheresis product, or other less common sources, such as umbilical cord blood. Red blood cells in blood can be lysed. Then the blood is caused to flow through the microfilter with small pores to keep the leukocytes.
• Another exemplary application is using the microfilter for chemo taxis applications. Membranes are used in the study of white blood cell reactions to toxins, to determine the natural immunity in whole blood.
• microfilters can be used to remove large emboli, platelet aggregates, and other debris.
• arrays of precision micro-pores can be fabricated in rolls of polymer resists in accordance with embodiments of the invention described above. Such arrays may be used for applications for which wafer-sized microfilters are not suitable. Examples of such applications include water filtration, kidney dialysis, etc.

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