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WO2022076697A1 - Dispositifs de séparation magnétique et procédés d'utilisation et de fabrication des dispositifs - Google Patents

Dispositifs de séparation magnétique et procédés d'utilisation et de fabrication des dispositifs Download PDF

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Publication number
WO2022076697A1
WO2022076697A1 PCT/US2021/053987 US2021053987W WO2022076697A1 WO 2022076697 A1 WO2022076697 A1 WO 2022076697A1 US 2021053987 W US2021053987 W US 2021053987W WO 2022076697 A1 WO2022076697 A1 WO 2022076697A1
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Prior art keywords
magnetic separation
separation device
magnetically
magnetic
soft material
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English (en)
Inventor
Erica BOETEFUER
Joshua BUSER
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Chip Diagnostics Inc
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Chip Diagnostics Inc
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications

Definitions

  • CTCs circulating tumor cells
  • pathogenic bacteria circulating microvesicles
  • exosomes exosomes
  • magnetophoresis in which immunomagnetically labeled targets are isolated from suspensions using strong and highly localized magnetic forces. Due to the lack of magnetic susceptibility of biological materials, magnetic sorting can be performed directly on unprocessed clinical samples (e.g., blood) and environmental samples (e.g., drinking water). Furthermore, strong forces can be applied without the need for a power supply or moving parts, making these devices well suited for use in practical settings outside of the laboratory.
  • Micropatterned magnetic field profiles have been engineered using lithographically defined current carrying wires and paramagnetic materials. Additionally, a number of bottom-up fabrication strategies have been developed to create strong magnetic forces. Microfluidic channels have been used in conjunction with patterned magnetic fields to bring targeted cells close to the high magnetic field gradients, to provide predictable flow velocities, and to minimize nonmagnetic retention.
  • magnetic separation devices can be useful in many applications, the current manufacturing process can limit the high throughput production of these devices. There is a need for magnetic separation devices that have improved sorting efficiencies, greater throughput, and/or easier manufacturing process.
  • a magnetic separation device comprising a magnetic separation filter encapsulated in an enclosed laminated structure, wherein the magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores.
  • the magnetically soft material comprises a nickel-iron alloy.
  • the magnetically soft material comprises Ni2oFeso.
  • the magnetic separation device further comprises a passivation layer adjacent the layer of magnetically soft material.
  • the passivation layer comprises nickel or gold.
  • the magnetic separation device comprises two or more magnetic separation filters. In some cases, the two or more magnetic separation filters are stacked together in the magnetic separation device.
  • the enclosed laminated structure is connected to a reservoir via one or more inlet ports.
  • the reservoir is configured to receive a suspension comprising a biological sample.
  • the reservoir is a syringe or microwell plate.
  • the reservoir is a 6, 12, 24, 48, 96, 384, or 1536 microwell plate.
  • each well in the microwell plate is connected to the magnetic separation filter.
  • the enclosed laminated structure is configured to prevent exposure of the magnetic separation filter to air when the inlet and outlet ports are closed.
  • the enclosed laminated structure is configured to prevent formation of a meniscus on the magnetic separation filter.
  • the enclosed laminated structure is configured to prevent oxidation of the magnetically soft material.
  • the magnetic separation device further comprises one or more inlet and outlet ports.
  • the one or more inlet and outlet ports are injection molded ports.
  • the one or more inlet and outlet ports are poly (methyl methacrylate) injection molded Luer lock ports.
  • a method for making the magnetic separation device disclosed herein comprising laminating a membrane roll onto a carrier substrate to form the magnetic separation filter encapsulated in the enclosed laminated structure, and wherein the magnetic separation filter comprises the layer of magnetically soft material having the plurality of pores.
  • a method of making a magnetic separation device comprising laminating a membrane roll onto a carrier substrate to form a magnetic separation filter encapsulated in an enclosed laminated membrane structure, and wherein said magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores.
  • the membrane roll is a track etched membrane roll.
  • the method further comprises magnetron sputtering of the magnetically soft material.
  • a method for using the magnetic separation device disclosed herein comprising: a) exposing the magnetic separation device to an external magnetic field; b) flowing a suspension comprising the magnetically tagged particles through an inlet port of the magnetic separation device; and c) capturing the magnetically tagged particles in the magnetic separation device.
  • a method for using a magnetic separation device comprising: a) exposing the magnetic separation device to an external magnetic field, wherein the magnetic separation device comprises a magnetic separation filter encapsulated in an enclosed laminated structure, wherein the magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores; b) flowing a suspension comprising a magnetically tagged particles through an inlet port of the magnetic separation device; and c) capturing the magnetically tagged particles in the magnetic separation device.
  • the method further comprises flowing a lysis reagent to the magnetic separation device, thereby contacting the captured magnetically tagged particles and releasing contents of the captured magnetically tagged particles.
  • the method further comprises removing the external magnetic field, thereby releasing the captured magnetically tagged particles.
  • the magnetically tagged particles comprises microorganisms, extracellular vesicles, cell-free DNAs or combinations thereof.
  • the microorganisms are selected from the group consisting of bacteria, viruses, or cells.
  • the cells comprise circulating tumor cells (CTCs).
  • the extracellular vesicles are selected from the group consisting of ectosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, exomeres, and bacterial outer membrane vesicles (OMVs).
  • the contents of the captured magnetically tagged particles are selected from the group consisting of proteins, nucleic acids, lipids, metabolites, and organelles.
  • FIGs. 1A and IB show a diagram representation (1 A) and a picture representation (IB) of an exemplary open system of the magnetic separation device.
  • FIGs. 2A and 2B show a diagram representation (2A) and a picture representation (2B) of an exemplary closed system of the magnetic separation device presented herein.
  • FIGs. 3A and 3B show conditions of the membrane for an exemplary open system (3 A) and closed system (3B).
  • FIG. 4 shows an exemplary material and assembly breakdown of the closed system magnetic separation device.
  • Magnetic separation device is used to refer to a device through which material flows through a magnetic separation filter, and which magnetically captures targeted objects.
  • the targeted objects may be magnetically tagged objects, such as, for example, cells, molecules, nucleic acids, proteins, etc.
  • the magnetic separation filter comprises a membrane having a plurality of pores, a layer comprising a magnetically soft material, and a passivation layer.
  • a magnetic separation filter comprises a magnetically soft material comprising a plurality of holes through which material may pass.
  • pore and “micropore” are used interchangeably to refer to channels that pass completely through the magnetic separation filter, i.e., continuous channels that pass from one surface of the filter to the opposite surface of the filter.
  • the layer of magnetically soft material in the magnetic separation device may comprise a material selected based on its magnetic properties.
  • magnetically soft material refers to a material which can become magnetized by a relatively low-strength, external magnetic field, e.g., by a magnet placed in close proximity to the material, that returns to a state of relatively low residual magnetism when the external magnetic field is removed.
  • the magnetically soft material is capable of having an induced magnetic field when an external magnetic field is applied.
  • the magnetically soft material may also be selected based on the magnetic remanence, i.e., the material’s ability to return to a nonmagnetic state when the external magnetic field is removed.
  • the magnetically soft material is selected from permalloys, which include alloys of nickel and iron.
  • the magnetically soft material is Ni2oFeso, an alloy which comprises 20% nickel and 80% iron (w/w).
  • the magnetic separation filter may comprise a passivation layer to protect the magnetically soft material from undesired interaction or reaction with fluids that the magnetic separation filter may come in contact with.
  • the passivation layer may protect the magnetically soft material from oxidation or prevent non-specific adsorption of biological substances to surfaces of the filter.
  • the passivation layer is comprised of a material chosen from minimally biologically active materials, such as, for example, gold or nickel.
  • the minimally biologically active material can be oxidation resistant. Other materials known to those skilled in the art capable of protecting the magnetically soft material from oxidation may be used.
  • the membrane is a material chosen from cellulosic, polymers, and metal oxide films. Examples of materials that may be used include, but are not limited to, paper, polycarbonate, polyester, nylon, and aluminum oxide. In at least one case, the membrane is polycarbonate.
  • the membrane is composed of a material capable of being ion track etched. Ion track-etching can be used to provide uniform pore sizes in the membrane material. Pores formed by ion track-etching are generally circular in shape and are typically randomly arranged in the film.
  • a magnetic separation filter comprising ion track-etched pores greater than 1 pm in diameter is referred to herein as a Track-Etched magnetic Micro-POre (TEMPO) filter, which are used in various cases and examples used throughout the present disclosure.
  • TEMPO Track-Etched magnetic Micro-POre
  • TENPO Track-Etched magnetic Nano-POre
  • TEMPO and TENPO filters differ only in the size of the pores, and, unless specifically stated, the description of TEMPO filters herein can be equally applied to TENPO filters.
  • the terms “microfluidic” and “nanofluidic,” as used herein, differ only in scale and all references to microfluidic are applicable to nanofluidic devices, unless stated otherwise. Other methods of forming pores within membranes known in the art can also be used.
  • the magnetic separation devices of the present invention are not intended to be limited to ion track- etched devices and one skilled in the art will recognize that unless otherwise specified, cases which refer to TEMPO or TENPO filters may include membrane-based magnetic separation filters formed by other methods. In at least one case, the membrane is ion track-etched polycarbonate.
  • the magnetically soft material is formed on the membrane by thermal evaporation, sputtering, chemical vapor deposition.
  • the layer of magnetically soft material formed on the membrane may have a thickness ranging from about 50 nm to about 1 pm, such as from about 50 nm to about 200 nm. In some cases, the layer of magnetically soft material may have a thickness of about 20 nm to about 2,000 nm. In some cases, the layer of magnetically soft material may have a thickness of at least about 20 nm. In some cases, the layer of magnetically soft material may have a thickness of at most about 2,000 nm.
  • the layer of magnetically soft material may have a thickness of about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1,000 nm, about 20 nm to about 1,500 nm, about 20 nm to about 2,000 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000 nm, about 50 nm to about 1,500 nm, about 50 nm to about 2,000 nm, about 100 nm to about 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about 1,500 nm, about 100 nm to about 2,000 nm, about 200 nm, about 100 nm to about 500 nm, about
  • the layer of magnetically soft material may have a thickness of about 20 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 1,500 nm, or about 2,000 nm.
  • the layer of magnetically soft material is evaporated on the membrane to form a layer having a thickness of 200 nm.
  • the thickness of the magnetically soft material formed on the membrane may be limited by the technique used to deposit the material. The thickness should be sufficient to generate a magnetic field strong enough to capture the desired particles.
  • the membrane comprises a commercially available ion track-etched polycarbonate membrane.
  • the membrane is coated with a thin layer of magnetically soft material (e.g., permalloy) and a passivation layer of gold.
  • Polycarbonate membranes can be track-etched with pore sizes ranging from 15 nm to 100 pm over large areas (A > 10 cm 2 ) for little cost ( ⁇ $.05/cm 2 ).
  • the membranes are flexible and can be integrated into laminate sheet microfluidics patterned with laser micromachining. Due to the large size of the membranes (A > 1 cm 2 ), highly efficient isolation (S, > 10 4 ) can be achieved at extremely high flow rates ( > 10 mL/hr).
  • the hydrodynamic drag force Fd 67tpav, where p is the viscosity and v is the fluidic velocity, can be minimized by using columnar flow instead of flow that is in-plane with a 1 in 3 NdFeB magnet.
  • the cross sectional area of a vertical flow channel grows quadratically with the dimensions of the chip L 2 , rather than linearly as with lateral flow. This feature may allow large flow rates ⁇ I> to be obtained, while keeping the flow velocity v small and the chip compact. Utilizing this approach, efficient sorting can be achieved at very high flow rates ( ⁇ E> > 10 mL/hr). Close proximity of each particle (e.g., cells or exosomes) to the regions of strong magnetic force (4).
  • the magnetic trapping force F m must overcome the drag force Fd.
  • the drag force is proportional to the flow velocity of the fluid Fd oc v.
  • a finite element model was developed to simulate the magnetic trapping capability of a TENPO according to the present disclosure using Matlab and Ansoft.
  • the field strength B drops rapidly in distance from the edge of the nanopore, creating field gradients B that lead to strong magnetic forces Fm.
  • the magnetophoretic force Fm on an exosome as it passes through a nanopore is calculated by combining the results from the simulation from with a simplified model for the exosome.
  • MNPs magnetic nanoparticles
  • m P 9.27xl0' 3 mA* pm 2 and that each targeted exosome has n > 5 MNPs.
  • the Stoke's drag on the trapped exosome F 6npav is calculated, where p is the viscosity of serum or plasma, and we find that even for extremely high flow rates »100 mL/hr) the magnetic force greatly exceeds the drag force F m » Fa.
  • the capture of an exosome is determined solely by its translation to the pore's edge, which is a function of its initial radial position r, the radial magnetophoretic force Fr, and its flow velocity v z oc From this analysis, the following observations were made: 1. The capture rate generally decreases as flow rate increases, 2. The capture rate generally increases as the pore's diameter d decreases, and 3. Because the probability of capturing an exosome is a function of its initial radial position in the pore, the capture rate can be increased by placing multiple filters in series, allowing the target object multiple, independent chances to be captured.
  • the magnetic separation filter may comprise an unsupported layer of magnetically soft material.
  • the term “unsupported layer of magnetically soft material” refers to a self-supporting layer of magnetically soft material, i.e., the layer of magnetically soft material does not need to be formed on another layer to provide support.
  • the layer of magnetically soft material may have a thickness sufficient to provide the necessary strength to support itself within a magnetic separation device and to endure the pressure generated by flow through the device.
  • the unsupported layer of magnetically soft material is not formed on a membrane.
  • the magnetic separation filter comprises pores at a pore density of at least 1000 pores/mm 2 , such as, for example, at least 1500 pores/mm 2 , at least 2000 pores/mm 2 , or more. In some cases, the magnetic separation filter comprises pores at a pore density of at least 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 pores/mm 2 .
  • pore density may be selected to reduce the potential for overlap.
  • MagNET filters pore density may be increased without overlap of the pores.
  • the pores may have an average diameter ranging from about 15 nm to about 100 pm, such as, for example, from about 100 nm to about 50 pm, from about 500 nm to about 50 pm, from about 500 nm to about 25 pm, or from about 500 nm to about 10 pm. In some cases, the pores may have an average diameter of about 50 nm to about 50,000 nm. In some cases, the pores may have an average diameter of at least about 50 nm. In some cases, the pores may have an average diameter of at most about 50,000 nm.
  • the pores may have an average diameter of about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 500 nm, about 50 nm to about 1,000 nm, about 50 nm to about 5,000 nm, about 50 nm to about 10,000 nm, about 50 nm to about 25,000 nm, about 50 nm to about 50,000 nm, about 100 nm to about 200 nm, about 100 nm to about 500 nm, about 100 nm to about 1,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 10,000 nm, about 100 nm to about 25,000 nm, about 100 nm to about 50,000 nm, about 200 nm to about 500 nm, about 200 nm to about 1,000 nm, about 200 nm to about 5,000 nm, about 200 nm to about 10,000 nm, about 200 nm to about 1,000 nm
  • the pores may have an average diameter of about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 5,000 nm, about 10,000 nm, about 25,000 nm, or about 50,000 nm. In at least one case, the pores have an average diameter less than about 50 pm, such as, for example, less than about 25 pm, less than 10 pm, less than about 5 pm, less than about 2 pm, or less than about 1 pm. As one skilled in the art would recognize, the size of the pores may be selected based on the size of the objects being separated.
  • the size of the pores is selected such that the pores are large enough not to trap the targeted objects, or nonspecifically trap nontargeted objects based on size, but small enough to expose the objects to the greatest magnetic field gradient possible.
  • the pore size may be 4 pm in diameter.
  • the pore size is about 2 to 5 times the size of the target object.
  • the pore size can range from about 50 nm to 1 pm. Larger pore sizes may also be used depending on the size of the target particles or to prevent co-purification of other particles present in the sample caused by trapping due to particle size.
  • a pore size of 500 nm could trap particles in a sample greater than 500 nm based on the inability of those particles to pass through the pores.
  • additional filters can be used in series.
  • the pore sizes of TEMPO/TENPO filters can be significantly smaller than the pore sizes of MagNET filters. Iontrack etching currently allows for the formation of pore sizes as small as 15 nm, whereas currently electroforming technology allows for the formation of pore sizes on the scale of a few micrometers.
  • the pores within the membrane may have any cross-sectional shape, such as, for example, circular, oval, rectangular, square, or other polygonal shape.
  • the pores are generally circular in shape.
  • the pore shape influences the magnetic field gradient.
  • the pores have a symmetrical geometry.
  • the pores have a circular cross-section. Without wishing to be limited by theory, it is believed that a circular cross-section provides the most uniform magnetic field gradient.
  • the shape of the pores may affect the efficiency of the magnetic separation filter. As discussed below, capture of magnetic particles occurs when the particle enters the magnetic field of the magnetic separation filter, which is strongest at the edge of the filter.
  • An elongated pore such as an oval or rectangular pore may increase the edge density of the pores in the device by increasing the effective length of the edge for a given number of pores, as compared to circular or square pores. Therefore, in accordance with at least one case, the pore shape may be selected to maximize the edge density of the magnetic separation filter.
  • the magnetic separation filters according to the present invention may allow for much greater flow rates than other available separation devices, such as microfluidic devices, which run at 1 ml/h.
  • TEMPO/TENPO filters have been prepared with a throughput up to about 40 ml/h with high enrichment.
  • the inventors have made MagNET filters having a throughput of 180 ml/h with an enrichment greater than 10 3 .
  • the magnetic separation devices according to cases of the present invention may be flexible. Flexibility of the magnetic separation device can be beneficial in the construction of microfluidic devices, for example in high throughput roll-to-roll fabrication processes. Rigid devices, such as those constructed of silicon, may be difficult to manipulate within the confines of small structures, such as those found in microfluidic devices.
  • the microfluidic/nanofluidic device comprises at least one lateral flow channel and at least one vertical flow magnetic separation filter.
  • the vertical flow magnetic separation filter such as, for example, a TEMPO/TENPO filter or MagNET filter, which comprises a membrane having a plurality of pores, a layer of magnetically soft material disposed on the membrane, and a passivation layer disposed on the layer of magnetically soft material.
  • the microfluidic/nanofluidic device may comprise any known structural or functional element.
  • the microfluidic/nanofluidic device can be modular, including the vertical flow magnetic separation filter.
  • the microfluidic/nanofluidic device comprises a plurality of vertical flow magnetic separation filters. Because each additional vertical flow magnetic separation filter increases the enrichment, one of ordinary skill in the art would recognize that the number of vertical flow magnetic separation filters can be selected to achieve the desired level of enrichment.
  • the microfluidic/nanofluidic device comprises from 2 to 10 vertical flow magnetic separation filters, such as, for example, from 2 to 5. In other cases, the microfluidic/nanofluidic device could contain more than 10 vertical flow magnetic separation filters.
  • the plurality of vertical flow magnetic separation filters can be arranged in series.
  • each of the plurality of vertical flow magnetic separation filters has a membrane containing pores and a pore density that are similar.
  • each of the vertical flow magnetic separation filters may have different pore sizes and/or pore densities.
  • the microfluidic/nanofluidic device may comprise a plurality of TEMPO/TENPO filters or a plurality of MagNET filters. In other cases, the microfluidic/nanofluidic device may combine at least one TEMPO/TENPO filter and at least one MagNET filter.
  • the microfluidic/nanofluidic device may comprise a flow converter for redirecting the lateral flow in the at least one lateral flow channel to vertical flow in the at least one vertical flow magnetic separation filter.
  • the flow converter may comprise, for example, a plurality of pathways through which fluid can pass from the lateral flow channel to the vertical flow magnetic separation filter.
  • Each of the plurality of pathways may be of similar length, such that fluid passing through the microfluidic/nanofluidic device will have the same residence time regardless of the path through which the fluid flows.
  • the flow converter comprises a symmetric branched geometry.
  • microfluidic/fluidic device can comprise an acrylic substrate, a lower 200 pm mylar layer, a TEMPO/TENPO filter, a flow converter comprising a layer of 50 pm mylar film having 16 regularly spaced holes and a layer of 200 pm mylar film having a symmetric branched geometry in fluidic communication with the 16 regularly spaced holes and fed by a lateral flow channel, and a top layer of 50 pm mylar film.
  • Another aspect of the present disclosure relates to a method for separating magnetically tagged particles in a microfluidic/nanofluidic device.
  • the method comprises exposing a vertical flow magnetic separation filter to an external magnetic field to induce a magnetic field gradient within pores of a membrane in the vertical flow magnetic separation filter, flowing a suspension comprising magnetically tagged particles through a lateral flow channel in a microfluidic/nanofluidic device, capturing the magnetically tagged particles in the pores of the vertical flow magnetic separation filter, removing the external magnetic field, and releasing the captured magnetically tagged particles.
  • a method for using the magnetic separation device comprising: exposing the magnetic separation device to an external magnetic field, wherein the magnetic separation device comprises a magnetic separation filter encapsulated in an enclosed laminated structure, wherein the magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores; flowing a suspension comprising the magnetically tagged particles through an inlet port of the magnetic separation device; capturing the magnetically tagged particles in the magnetic separation device; and flowing a lysis reagent through the inlet port of the magnetic separation device, thereby contacting the captured magnetically tagged particles and releasing contents of the captured magnetically tagged particles.
  • the magnetically tagged particles can be microorganisms (e.g., bacteria, viruses, cells such as circulating tumor cells (CTCs)) or extracellular vesicles (e.g., exosomes, bacterial outer membrane vesicles (OMVs)).
  • CTCs circulating tumor cells
  • OMVs extracellular vesicles
  • the contents of the captured magnetically tagged particles can be proteins, nucleic acids (e.g., DNAs or RNAs), lipids, metabolites, or organelles from the captured magnetically tagged particles.
  • the magnetically soft material and the passivation layer can be formed on the membrane using any technique known in the art.
  • the materials may be deposited by thermal evaporation, sputtering, chemical vapor deposition, electroplating, etc.
  • the unsupported layer of magnetically soft material is produced by electroforming the magnetically soft material.
  • An electroformed nickel-iron alloy filter is referred to herein as a MAgnetic Nickel-iron Electroformed Trap (MagNET) filter.
  • MagNET MAgnetic Nickel-iron Electroformed Trap
  • methods and materials other than electroforming and nickel-iron alloys may be used to prepare magnetic separation filters comprising an unsupported layer of magnetically soft material. Therefore, embodiments which refer to MagNET filters may include magnetic separation filters having an unsupported layer of magnetically soft material formed by other methods.
  • the layer of magnetically soft material in a MagNET filter is formed by electroforming the layer on a mold.
  • the mold may comprise any material on which the magnetically soft material may be electroformed and separated.
  • the layer of magnetically soft material may be electroformed and mechanically removed from the mold, such as by peeling the layer from the mold.
  • the layer of magnetically soft material may be removed by etching the mold away from the electroformed layer.
  • the layer of magnetically soft material is electroformed on a mold and mechanically removed, enabling the mold to be reused to form additional layers.
  • the mold is made of copper.
  • the mold may comprise a release layer to improve the release properties of the electroformed layer from the mold.
  • a non-limiting example of a release layer formed on a copper mold is titanium.
  • the mold may comprise pillars or protrusions that correspond to the pores when the layer of magnetically soft material is electroformed on the mold.
  • the pillars or protrusions may be made of the same or different material as the mold.
  • the pillars or protrusions are formed of a photoresist.
  • the photoresist may be patterned using photolithography, for example.
  • the photoresist is a positive photoresist.
  • the sides of the pillars or protrusions may be tapered to improve release of the electroformed layer from the mold.
  • the degree of taper is selected based on the desired thickness of the electroformed layer, the shape of the pores, and/or the size of the size of the pores.
  • the layer of magnetically soft material in the MagNET filter has a thickness ranging from about 3 pm to about 40 pm, such as, for example from about 5 pm to about 25 pm. Thicker or thinner layers may also be used. The thickness may be limited by the manner in which the layer of magnetically soft material is formed.
  • a layer that is too thin may not be able to be removed from a mold, whereas a layer that is too thick may damage pillars or protrusions on the mold when it is removed.
  • the thickness may also depend on the desired properties of the MagNET filter. Without wishing to be bound by theory, it is believed that MagNET can capture magnetic particles at the top and bottom of each pore.
  • the magnetically soft material in the MagNET filters may have a surface passivation layer, such as an inert material like gold or nickel.
  • the pores of the MagNET filters may be selected from any desired shape. Because the molds can be made using techniques such as photolithography, there is no limit to the shape that may be created.
  • the pores may have circular, square, triangular, oval, or rectangular shapes. Other, more complex shapes are also possible.
  • the shape of the pore may be tailored to match the shape of the desired target particles. If the target particles are cell clusters, the pores may have a clover shape, for example, or another shape to maximize the potential for trapping the particles in the magnetic separation filter.
  • the magnetic separation device can be used for the diagnosis of or risk-profiling for conditions or diseases, such as cancer or brain injury, by capturing magnetically tagged particles, such as extracellular vesicles (e.g., exosomes).
  • Extracellular vesicles e.g., exosomes
  • proteins biomarkers as well as fragments of mRNA, miRNA, and DNA from their mother cells.
  • biomarkers can be used to determine whether a subject has a specific condition. For example, a TEMPO/TENPO filter as described above may be used to isolate one or more exosomes.
  • Exosomal biomarkers may be tagged with magnetic nanoparticles (MNPs), such as iron oxide nanoparticles or any other magnetic material known in the art, and trapped by a magnetic separation filter according to an example disclosed herein (e.g., a TEMPO/TENPO filter).
  • MNPs magnetic nanoparticles
  • the extracellular vesicles may be incubated with a cocktail of biotinylated antibodies and subsequently incubated with anti -biotin MNPs.
  • Extracellular vesicles (e.g., exosomes) trapped by the magnetic separation filter may be evaluated by analyzing the nucleic acids or proteins extracted from the extracellular vesicles (e.g., exosomes), e.g., by using qPCR.
  • multiple biomarkers may be used to enable the method to detect more than one condition or disease.
  • Conditions or diseases that may detected include any condition or disease which can be detected by biomarkers contained in an exosome, such as cancer (e.g., pancreatic cancer, lung cancer, prostate cancer, breast cancer, bladder cancer, liver cancer, glioblastomas), addiction, tuberculosis, brain injuries (including ischemic brain injury and traumatic brain injury), or infectious disease (e.g., tuberculosis, HIV, COVID-19).
  • cancer e.g., pancreatic cancer, lung cancer, prostate cancer, breast cancer, bladder cancer, liver cancer, glioblastomas
  • addiction e.g., tuberculosis
  • brain injuries including ischemic brain injury and traumatic brain injury
  • infectious disease e.g., tuberculosis, HIV, COVID-19
  • brain-derived exosomes have been found in the bloodstream after brain injury.
  • the method according to the present invention can be used to isolate and identify these exosomes.
  • the exosomes may be isolated from samples including blood/
  • the magnetic separation device can comprise a closed system.
  • the comparison of the original open system (FIGs. 1A and IB) and an exemplary closed system (FIGs. 2A and 2B) is shown here.
  • similar magnetic separation devices feature a larger, open reservoir, which has several negative impacts.
  • the open system 100 comprises an open reservoir 110, which receives the sample and/or reagents, and can expose the sample and reagents to air, thereby leading to a high potential for evaporation and/or contamination.
  • the open reservoir 110 can cause the formation of a meniscus 120 on the magnetic capture filter membrane, which can allow air to enter the middle of the reservoir area before the fluid in the meniscus 120 (around the edges of the reservoir) is pulled through, and requires a larger sample volume.
  • the open reservoir 110 can have a higher run-to-run variability, which may require manual intervention during operation.
  • membrane damage e.g., flaking of the metal
  • clogging of the system during the lysis step can reduce the efficacy of the open system 100.
  • the use of a closed system 200 can overcome the above potential deficiencies of the open system 100.
  • the closed system 200 can comprise an inlet port 210, fed by a reservoir 230 (e.g., syringe can be used as the reservoir, as shown in FIG. 2B), and two outlet ports 220.
  • the top of the magnetic capture zone can be fed by a fluidic manifold similar to the design used on the bottom of the membranes.
  • the protocol can be automated such that the reservoir 230 is not allowed to run empty, thereby preventing air from entering the system.
  • the closed system 200 can also allow compatibility with automation, improving reliability and/or reproducibility. Accordingly, the meniscus can be limited to a smaller reservoir that is not allowed to run dry.
  • the use of a smaller surface area and a taller column or a cap with a one-way valve can reduce rate of evaporation.
  • switching from the open system 100 to a closed system 200 can eliminate the membrane damage and/or clogging during the lysis step.
  • the open system sustained membrane damage during lysis, which can be caused by oxidation and/or NiFe exposure through gold passivation layer.
  • Such membrane damage of the open system can cause oxidation and/or pore damage that can clog the device.
  • membrane damage can be caused by oxidation and/or NiFe exposure through gold passivation layer.
  • the closed system in FIG. 3B survived the lysis step without membrane damage, potentially due to oxygen exclusion and/or improved membrane integrity. Accordingly, the closed system can provide optimized lysis and/or increased yield, and thus simplifying the purification steps (e.g., for miRNA purification).
  • the magnetic separation device can have an improved fluid interface.
  • the open system 100 included a punched polydimethylsiloxane (PDMS) outlet port 130 that required PDMS casting, punching, and plasma bonding, which can increase the difficulty/complexity of the manufacturing process.
  • the closed system 200 can comprise injection-molded inlet port 210 and outlet ports 220 (e.g., poly(methyl methacrylate) (PMMA) Luer lock ports) and/or the same adhesive (3M 444 adhesive) that is in the laminated structure of the chip.
  • PMMA poly(methyl methacrylate)
  • the closed system can also improve reproducibility. A detailed material and assembly breakdown of the closed system magnetic separation device 200 can be found in FIG.
  • the magnetic separation device can be manufactured by raster printing a polymer sheet carrier with low-tack adhesive using a non-adhesive pattern to reduce bonding strength and/or prevent tearing of the membrane when it is removed later in the process.
  • Track etched membrane rolls (e.g., Cytiva Nuclepore) can be laminated onto the carrier, and the assembly cut into squares, and mounted to a fixture for magnetron sputtering of nickel-iron alloy followed by a gold passivation layer. After metal coating, a circular kiss-cut die cutter can cut through the membrane portion of the laminate, and the waste membrane can be removed. Layers of the chip can be held in place using a vacuum plated alignment system for assembly.
  • the magnetic separation filter membrane can be manufactured, for example, coated with magnetic material and subsequently assembled into a laminated device, in a roll format or disc format.
  • the magnetic device manufactured with a roll format can be advantageous in comparison to the disk format, because it can enable the assembly of multiple (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) chips simultaneously and/or it can be compatible with roll-to-roll processes for further scaling of the manufacturing process.
  • Membranes of the high-throughput TENPO in a roll format can be processed and mounted into a fixture. Membranes can be cut into rectangular sections to cover the bottom of a 96-well plate. A 96-well plate can be prepared with a hole protruding through the bottom of each well. Metal coated membranes and double-sided adhesives can be stacked to create independent capture regions for each well. After the capture region, fluidic channels composed of doublesided adhesives and plastic sheets can connect the outlet of each capture region to a common two fluidic ports (“outlet 1” and “outlet 2”).
  • the manufacturing process can be adapted to produce a multiplexed version of the magnetic device, which can be used to process multiple samples simultaneously, i.e., can handle more than one pulldown on more than one sample at a time.
  • the multiplexed version of the magnetic device can process multiple patient samples with established targeted assays such as pancreatic cancer, traumatic brain injury, or lung cancer.
  • the multiplexed version of the magnetic device can isolate extracellular vesicles (EVs) originating from multiple organs from a single patient blood sample (e.g., multiorgan scan).
  • the improved manufacturing process can decrease the footprint of the device, add alignment features, and make layer size uniform to improve manufacturability.
  • the multiplexed version can provide multi-well compatibility.
  • the magnetic separation device e.g., TENPO
  • the high-throughput TENPO device can be used in a multi-step process: 1) magnetic labeling: samples can be incubated with antibodies and/or magnetic nanoparticles to magnetically tag biological objects of interest (e.g., extracellular vesicle or EV); 2) magnetically tagged samples can run through the multiplexed TENPO device by extracting from “outlet 1”, while a magnetic field is applied to the device.
  • biological objects of interest e.g., extracellular vesicle or EV
  • Targeted biological objects can be captured in each well and rinsed as needed; 3) buffer can be added to “outlet 2” and extracted from “outlet 1”, clearing the outlet channels of any remaining sample; 4) lysis reagent can be added to “outlet 2” and extracted from “outlet 1”, loading the outlet channel network with lysis reagent; 5) backpressure can be applied to both “outlet 1” and “outlet 2”, causing lysis reagent flow back into each capture reagent, and causing captured EVs to lyse and the contents of each well’s capture region to backflow into the well into which the sample was added; 6) lysate can be removed from each individual well and analyzed.
  • buffer can be added to “outlet 2” and extracted from “outlet 1”, clearing the outlet channels of any remaining sample
  • lysis reagent can be added to “outlet 2” and extracted from “outlet 1”, loading the outlet channel network with lysis reagent; 5) backpressure can be applied to both “outlet 1” and “outlet 2”,
  • Embodiment E A magnetic separation device, comprising a magnetic separation filter encapsulated in an enclosed laminated structure, wherein the magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores.
  • Embodiment 2 The magnetic separation device of embodiment 1, wherein the magnetically soft material comprises a nickel-iron alloy.
  • Embodiment 3 The magnetic separation device of embodiment 1 or 2, wherein the magnetically soft material comprises Ni2oFeso.
  • Embodiment 4 The magnetic separation device of any one of embodiments 1-3, further comprising a passivation layer adjacent the layer of magnetically soft material.
  • Embodiment 5 The magnetic separation device of embodiment 4, wherein the passivation layer comprises nickel or gold.
  • Embodiment 6 The magnetic separation device of any one of embodiments 1-5, comprising two or more magnetic separation filters.
  • Embodiment 7 The magnetic separation device of embodiment 6, wherein the two or more magnetic separation filters are stacked together in the magnetic separation device.
  • Embodiment 8 The magnetic separation device of any one of embodiments 1-7, wherein the enclosed laminated structure is connected to a reservoir via one or more inlet ports.
  • Embodiment 9 The magnetic separation device of embodiment 8, wherein the reservoir is configured to receive a suspension comprising a biological sample.
  • Embodiment 10 The magnetic separation device of embodiment 8 or 9, wherein the reservoir is a syringe or microwell plate.
  • Embodiment 11 The magnetic separation device of embodiment 10, wherein the reservoir is a 6, 12, 24, 48, 96, 384, or 1536 microwell plate.
  • Embodiment 12 The magnetic separation device of embodiment 10 or 11, wherein each well in the microwell plate is connected to the magnetic separation filter.
  • Embodiment 13 The magnetic separation device of any one of embodiments 1-12, wherein the enclosed laminated structure is configured to prevent exposure of the magnetic separation filter to air when the inlet and outlet ports are closed.
  • Embodiment 14 The magnetic separation device of any one of embodiments 1-13, wherein the enclosed laminated structure is configured to prevent formation of a meniscus on the magnetic separation filter.
  • Embodiment 15 The magnetic separation device of any one of embodiments 1-14, wherein the enclosed laminated structure is configured to prevent oxidation of the magnetically soft material.
  • Embodiment 16 The magnetic separation device of any one of embodiments 1-15, further comprising one or more inlet and outlet ports.
  • Embodiment 17 The magnetic separation device of embodiment 16, wherein the one or more inlet and outlet ports are injection molded ports.
  • Embodiment 18 The magnetic separation device of embodiment 16 or 17, wherein the one or more inlet and outlet ports are poly(m ethyl methacrylate) injection molded Luer lock ports.
  • Embodiment 19 A method for making the magnetic separation device of any one of embodiments 1-18, comprising laminating a membrane roll onto a carrier substrate to form the magnetic separation filter encapsulated in the enclosed laminated structure, and wherein the magnetic separation filter comprises the layer of magnetically soft material having the plurality of pores.
  • Embodiment 20 A method of making a magnetic separation device, comprising laminating a membrane roll onto a carrier substrate to form a magnetic separation filter encapsulated in an enclosed laminated membrane structure, and wherein said magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores.
  • Embodiment 21 The method of embodiment 19 or 20, wherein the membrane roll is a track etched membrane roll.
  • Embodiment 22 The method of any one of embodiments 19-21, further comprising magnetron sputtering of the magnetically soft material.
  • Embodiment 23 A method for using the magnetic separation device of any one of embodiments 1-18, comprising: a) exposing the magnetic separation device to an external magnetic field; b) flowing a suspension comprising the magnetically tagged particles through an inlet port of the magnetic separation device; and c) capturing the magnetically tagged particles in the magnetic separation device.
  • Embodiment 24 A method for using a magnetic separation device, comprising: a) exposing the magnetic separation device to an external magnetic field, wherein the magnetic separation device comprises a magnetic separation filter encapsulated in an enclosed laminated structure, wherein the magnetic separation filter comprises a layer of magnetically soft material having a plurality of pores; b) flowing a suspension comprising a magnetically tagged particles through an inlet port of the magnetic separation device; and c) capturing the magnetically tagged particles in the magnetic separation device.
  • Embodiment 25 The method of embodiment 23 or 24, further comprising flowing a lysis reagent to the magnetic separation device, thereby contacting the captured magnetically tagged particles and releasing contents of the captured magnetically tagged particles.
  • Embodiment 26 The method of any one of embodiments 23-25, further comprising removing the external magnetic field, thereby releasing the captured magnetically tagged particles.
  • Embodiment 27 The method of any one of embodiments 23-26, wherein the magnetically tagged particles comprises microorganisms, extracellular vesicles, cell-free DNAs or a combination thereof.
  • Embodiment 28 The method of embodiment 27, wherein the microorganisms are selected from the group consisting of bacteria, viruses, or cells.
  • Embodiment 29 The method of embodiment 28, wherein the cells comprise circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • Embodiment 30 The method of embodiment 27, wherein the extracellular vesicles are selected from the group consisting of ectosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, exomeres, and bacterial outer membrane vesicles (OMVs).
  • Embodiment 31 The method of any one of embodiments 25-30, wherein the contents of the captured magnetically tagged particles are selected from the group consisting of proteins, nucleic acids, lipids, metabolites, and organelles.
  • Pancreatic cancer is the fourth most common cause of cancer related death in the United States, with a five year survival rate of only 8%. Because pancreatic tumor cells are localized in difficult to access parts of the body, molecular measurements currently rely on invasive procedures (i.e., biopsy) which severely limit their practical diagnostic use. Nano-scale vesicles that originate from tumor/injured cells and which can be found circulating in the blood (e.g., exosomes) have been discovered to contain a wealth of proteomic and genetic information to monitor cancer progression, metastasis, and drug efficacy.
  • exosomes as biomarkers to improve patient care has been limited by fundamental technical challenges that stem from extreme scarcity and the small size of tumor- derived exosomes (30 nm-200 nm) and the extensive sample preparation (>24 hr) required prior to measurement.
  • exosomes were detected using a TEMPO filters in accordance with embodiments of the present invention, which combined the benefits of nanoscale sorting with extremely fast flow rates ( ⁇ 1 hr assay time).
  • the unbiased exosome detection achieved >5x yield compared to the conventional technique (ultracentrifugation).
  • LDA linear discriminant analysis
  • different groups of mice were classified (cancer vs. healthy). And more importantly, it was possible to distinguish pre-cancer mice from healthy mice.
  • Serum was magnetically labeled with anti-biotin magnetic nanoparticles and isolated using a TEMPO filter according to an embodiment of the present invention.
  • the RNA was extracted from the exosomes and amplified using qPCR. The cells and exosomes were positively correlated and it was possible to distinguish pre-cancer mice from healthy mice.
  • Exosomes were labeled in media from MiaPaCa2 cells with 50 nm iron oxide magnetic nanoparticles (Miltenyi Biotec) using a cocktail of the pan- exosome markers, CD81, CD9, and CD63.
  • exosomes were also fixed directly on the ExoTENPO nanopores after capture, and imaged using scanning electron microscopy (SEM) (University of Pennsylvania School of Medicine, Electron Microscopy Resource Laboratory). Objects were observed with a morphology consistent with exosomes.
  • SEM scanning electron microscopy
  • a model system which consisted of 12 ml of media from a cultured pancreatic cancer cell line (BxPC3) spiked into 3 ml of healthy human plasma was used. From this model system exosomes were isolated using a cocktail of pan exosome markers (CD63, CD9, CD81) as well as a tumor-specific markers, including EpCAM and Intpi.
  • LDA vectors LDA healthy, LDA cancer
  • Traumatic brain injury occurs in approximately 2.5 million people each year. Although it is a very common worldwide incident, the lack of molecular marker based diagnostic tools complicates clinical decision for monitoring and treatment of patients. An accurate assessment of the incident is crucial especially when the TBI patients sustain a secondary injury that can lead to a long-term physical, emotional, and behavioral disability.
  • imaging technologies such as computerized tomography (CT) scans and magnetic resonance imaging (MRI) can be used for severe TBI, but mild TBI (mTBI) diagnostics, which comprise of 70-90% of the TBI cases, are currently limited to patient reports and clinical symptoms, which do not provide an objective assessment.
  • exosomes are isolated using a bulky ultracentrifuge, which causes high loss, low purity, and long assay time. Due to these limitations, downstream analysis of exosomes is not practical and extremely difficult to achieve a reliable, meaningful result.
  • small RNA sequencing on exosomes isolated using an ExoTENPO chip achieved >5x yield, high purity (90%), and extremely rapid (>10 ml/hr) assay time. This experiment focused on discovering brain-derived exosomal miRNAs that were differentially expressed after mTBI using blast- induced injured mice.
  • the ExoTENPO chip was used to isolate exosomes based on their glutamate receptor 1/2 (GluRl/2) expression to profile brain-derived exosomes. It was discovered that exosomal miRNAs were differentially expressed after mTBI. A subset of these exosomal miRNAs were used to diagnose mTBI mice, achieving 100% sensitivity and 100% specificity.
  • GluRl/2 glutamate receptor 1/2
  • DLS dynamic light scattering
  • the isolate from the ExoTENPO chip showed a major peak at 141.8 nm, which was in the range of exosome size (30-200 nm). Scanning electron microscopy (SEM) was also performed in order to show that exosomes were captured at the edge of the pores of the chip. It was observed that 150-200 nm exosomes were captured at the edge of the pores.
  • mouse plasma was used for biomarker discovery. First, mouse plasma was run through the ExoTENPO chip. The chip allowed for specific enrichment of brain-derived exosomes by targeting the exosomes using an anti-glutamate receptor 1/2 (GluR2) antibody (biotin).
  • GluR2 anti-glutamate receptor 1/2
  • the biotinylated antibody was incubated with anti-biotin microbeads, which were magnetic iron oxide nanoparticles. As the plasma flowed through the chip, the labeled brain-derived exosomes were captured on edge of the pores of the chip. After exosome capture, the total exosomal RNA was isolated by lysing on the chip. Then, a small RNA library prep set (BioLabs) was used for RNA sequencing. Using the prepared samples, an RNA sequencer (Illumina) was run and the RNA sequencing data was evaluated using quantitative polymerase chain reaction (qPCR).
  • qPCR quantitative polymerase chain reaction
  • RNA sequencing data showed that 565 miRNAs were expressed by brain-derived exosomes from mice. Exosomal miRNAs were sequenced from two groups, control and injured mice. Healthy mice without injury were used as a control, and blast-induced injury was performed to mimic mTBI.
  • miRNAs there were 128 miRNAs that had raw counts more than 50. As expected, there were some miRNAs that were differentially expressed from the two groups and some that were similar to each other. The composition of brain-derived exosomal miRNAs expressed by control mice and those expressed by injured mice was also observed. The top 3 most abundant miRNAs were the same (miR-486b-5p, miR-486a-5p, let-7i-5p) between two the groups.
  • KEGG Kyoto encyclopedia of genes and genomes pathway analysis was performed to find a statistically significant pathways that are related to brain.
  • miR-21a-5p was chosen as the last marker based on the findings that showed it alleviates secondary blood-brain barrier damage after TBI and apoptosis of cortical neurons.
  • expression level of individual miRNA markers was obtained. There was a positive correlation between the expression level from qPCR and the normalized read count from RNA sequencing, with some variance. Then, heat maps were generated to observe a pattern for the expression level of the selected miRNA panel from different groups (injured, control). The patterns were different, but there were no miRNAs that were upregulated or downregulated in each individual mouse from one group.
  • mTBI diagnosis was performed on mice using the panel of miRNA markers that were validated using qPCR. Using the whole panel of miRNA markers, we were able to achieve 100% sensitivity and 100% specificity. In order to analyze the pattern for diagnosis, linear discriminant analysis (LDA) was used.
  • LDA linear discriminant analysis
  • RNA sequencing was used to make a library. RNA was isolated on chip using Total Exosomal RNA Isolation Kit (Life Technologies). [084] Then, the RNA amount was measured using Qubit (Life Technologies) and as recommended by the protocol, the samples with more than 100 ng of RNA were selected for usage. Then, quality control check was performed on a BioAnalyzer using a DNA 1000 chip. For size selection, AMPure XP beads were used (Beckman Coulter). 140-150 bp sizes were selected using the beads and the sizes were confirmed by the BioAnalyzer using High Sensitivity Chip. A NextSeq 500/550 kit (FC-404-2005, Illumina) was used for RNA sequencing.
  • BD VacutainerTM sodium citrate coated blood collection tubes
  • Peripheral whole blood was obtained from PDA patients with advanced pancreatic cancer and from healthy age- and gender-matched controls at the University of Pennsylvania Health System. All patients and healthy donors provided written informed consent for blood donation on approved institutional protocols.
  • Whole blood was drawn in either EDTA (Fisher Scientific), Streck Cell-Free DNA BCT® (Streck), or gel serum separation tubes (Fisher Scientific). Plasma and serum were isolated using the following procedures. Within 3 hours of blood draw for EDTA and within 12 hours of blood draw for Streck, tubes were centrifuged at 1600g for 10 minutes at room temperature with the break off.
  • plasma was transferred to a fresh 15 ml centrifuge tube without disturbing the cellular layer and centrifuged at 3000g for 10 minutes (EDTA) or 4122g for 15 minutes (Streck) at room temperature with the break off; this step was repeated with a fresh 15 ml centrifuge tube.
  • plasma was transferred to a fresh 15 ml centrifuge tube, gently mixed, and transferred in 1 ml aliquots to centrifuge tubes and either processed fresh for exosomal RNA or stored immediately at -80°C for future use.
  • Gel serum separation tubes were stored at room temperature for 30 minutes after blood draw. Within 2 hours of blood draw, serum tubes were centrifuged at 1000 g for 15 minutes at room temperature.
  • serum was transferred in 1 ml aliquots to cryovials and either processed fresh for exosomal RNA or stored immediately at -80°C for future use.
  • Mouse cell lines PD7591, PD483, PD6910 were generated from pancreatic tumor tissue isolated from Pdxl-cre, Kras LSL ' G12D , p53 L/+ , Rosa YFP/YFP (KPCY) mice (Rhim et al Cell 2012). They were cultured in pancreatic ductal epithelium media as previously described (Schreiber, F. S. et al. Successful growth and characterization of mouse pancreatic ductal cells: functional properties of the Ki-RASG12V oncogene). All human cell lines were cultured in media recommended by ATCC.
  • Anti-biotin ultrapure microbeads (Miltenyi Biotec) and biotinylated antibodies were used for magnetic labeling.
  • biotin anti-CD9 antibody BioLegend
  • biotin anti-CD81 antibody BioLegend
  • biotin anti-human CD9 antibody eBioscience
  • biotin anti-human CD63 antibody BioLegend
  • biotin anti-CD81 antibody custom made from BioLegend
  • Total exosome RNA & protein isolation kit (Life Technologies) was used for RNA extraction from isolated exosomes.
  • denaturing solution was added to the chip and the chip was incubated for 5 mins on ice. Then, the lysed solution was taken off chip for acid-phenol separation and washing steps using a spin column. The exosomal RNA was eluted in a small volume ( ⁇ 30 pl) and it was stored at -80C or processed immediately for further analysis.
  • Exosomal DNA Isolation [098] Exosomal DNA was isolated using QUIAamp DNA mini kit (Qiagen). Lysis buffer was directly added on chip and the chip was incubated at 56 C for 10 mins. Then, the lysed solution was taken off chip for the rest of the steps. The exosomal DNA was eluted in a small volume ( ⁇ 30 pl) and it was stored at -20C or -80C until usage.
  • RT-PCR was first performed using exosomal RNA.
  • PrimeScript RT Reagent Kit (Clontech) was used for RT-PCR.
  • the exosomal RNA was mixed with reagents and the sample was in a T100 Thermal Cycler (Bio Rad) followed by the company's protocol.
  • the size of the exosomal RNA and DNA was measured using a BioAnalyzer.
  • Exosomal RNA was measured in BioAnalyzer using the Agilent RNA Pico chip at the NAPCore Facility at the Children's Hospital of Philadelphia.
  • Exosomal DNA was measured in BioAnalyzer using the Agilent High Sensitivity DNA chip at the same facility.
  • the amount and concentration of the exosomal RNA and DNA were measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and the Qubit ddDNA Assay kit (Thermo Fisher Scientific) respectively.
  • Immunomagnetic sorting is a technique to selectively isolate rare magnetically-tagged cells from heterogeneous suspensions— yet current devices fail to provide high enrichment (Q for clinically relevant volumes (>30 mL) and turnaround times ( ⁇ 30 min).
  • Rare cells such as circulating tumor cells (CTCs) are present in concentrations of 1 - 10 2 in 10 mL of blood, requiring large samples of blood to be processed with high specificity to isolate these cells from the background of 10 5 leukocytes, IO 10 red blood cells, etc.
  • the filter was fabricated by electroplating permalloy (Ni2oFeso) onto a copper substrate patterned with an array of 15 pm tall, 30 pm diameter photoresist pillars (SPR220-7.0). Once the permalloy was plated to a thickness of 15 pm, the durable film was mechanically peeled from the mold to obtain a metal filter with 30 pm pores.
  • the edge of the pore creates a strong magnetic trap to capture magnetically tagged targets.
  • Copper molds can be replated multiple times, and filters can be reused without performance loss— offering a cost- effective fabrication strategy.
  • lithography allows higher pore density without overlap, design of traps in any shape, and filters with area >25 cm 2 .
  • Vertical fluid flow through the porous filter can process 30 mL of blood in 20 min with high capture rate on a compact chip— offering a key breakthrough to enable immunomagnetic sorting to be applied for rare cell detection in clinical diagnoses.
  • the MagNET filters were also reusable.

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Abstract

L'invention concerne des dispositifs de séparation magnétique pour séparer des objets biologiques marqués magnétiquement. Le dispositif de séparation magnétique comprend un filtre de séparation magnétique encapsulé dans une structure stratifiée fermée, le filtre de séparation magnétique comprenant une couche de matériau magnétiquement doux ayant une pluralité de pores. L'invention concerne également les procédés d'utilisation et de fabrication des dispositifs de séparation magnétique.
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