Particle separator system, material and method of use

Abstract

The present invention generally relates to the concentration of particle-containing samples, such as cells or biomolecules, in order to separate these particles in a medium and to separate the particle-removed medium. In some embodiments, the present invention generally relates to the isolation and / or concentration of nucleus from nucleus fragments and dead cells.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 489,985, filed March 13, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention generally relates to the concentration of particulate samples (e.g., cells or biomolecules) in order to separate these particles in a medium and to separate the medium from which the particles are removed. In some embodiments, the present invention generally relates to the separation and / or concentration of cell nuclei from nuclear debris and dead cells. Background Technology

[0004] Separating particles contained in a medium is an important step in many chemical and biological processes. In some methods, it may be necessary to simply separate the particles to facilitate their use or manipulation, while in others, it may be necessary to separate the particles from other particles present in the medium. Various devices have been developed to facilitate such particle separation. Furthermore, attempts have been made to develop devices that rely on the magnetism of the particles and their surrounding medium to separate target particles from heterogeneous particle populations.

[0005] When processing cells, it is often necessary to concentrate them by reducing the volume of the cell suspension. The most common method for cell concentration is to centrifuge the cells to form a precipitate and remove most of the medium. Centrifugation involves applying centrifugal force to separate particles from the solution based on particle size, shape, density, medium viscosity, and rotor speed. However, centrifugation is not ideal in some cases because it can damage or activate cells. For example, centrifugation of T cells can lead to cell activation. Additionally, when processing sparse or small samples, large-scale separation techniques such as centrifugation can be very wasteful or laborious and do not readily allow for sample fractionation. Furthermore, when the particles to be separated are fragile or unstable, such as when processing biological entities, achieving precise conditions that improve particle stability can be challenging.

[0006] When dealing with difficult-to-process tissues and samples, the cell nucleus has rapidly become one of the primary targets for single-cell research and development. Although processing cells should be simpler and more straightforward than processing the nucleus, the reality of processing tissues is often far more complex and difficult than expected.

[0007] Tissues vary in their cellular composition, extracellular matrix, and dissociation behavior during processing. Therefore, researchers find it increasingly difficult to establish well-validated and uniformly performed dissociation and purification protocols to produce reliable, high-quality single-cell suspensions. Alternatively, nuclear workflows overcome many of these challenges by not only providing more homogeneous and standardized starting materials for single-cell analysis but also removing some of the potential for gene expression changes associated with tissue dissociation and processing.

[0008] Unfortunately, cell nucleus preparation comes with its own challenges. Specifically, there's the issue of purifying extracted nuclei from dead cells and debris. Traditional methods remain cumbersome and time-consuming, resulting in low yields and purity. LeviCell offers a rapid, simple, and efficient method for purifying cell nuclei.

[0009] The apparatus and methods described herein address these problems by providing alternative methods for concentrating particles such as cells and cell nuclei, and for producing media that do not rely on the high mechanical forces required for particle removal during centrifugation. Summary of the Invention

[0010] The embodiments provided in the summary of this invention are merely illustrative and provide an overview of the alternative embodiments disclosed herein. The summary of this invention is illustrative and selective, does not limit the scope of any claims, does not provide the entire scope of the embodiments disclosed or contemplated herein, and should not be construed as limiting or restricting the scope of this disclosure or any claimed embodiments.

[0011] This article provides a method (“Method 1”) for extracting cell nuclei from intact cells, comprising: providing intact cells; and lysing the cells in a cell nucleus separation buffer; wherein the cell nucleus separation buffer contains wheat germ lectin (WGA).

[0012] This article provides a method (“Method 1a”) for reducing nuclear contamination from environmental RNA, including the isolation of cell nuclei in the presence of wheat germ lectin (WGA).

[0013] This article provides a method (“Method 2”) for separating target subcellular components, such as cell nuclei, from a sample containing target subcellular components and one or more contaminants, comprising: A sample comprising the target subcellular component, the contaminant, and the sample medium is loaded into a separation channel, wherein the sample optionally flows along the separation channel, and wherein the sample medium comprises: i) Paramagnetic compounds or ferrofluids; and ii) Separate particles (or microbeads); At least one magnet is used to apply a magnetic force to the sample in order to separate the target subcellular component from other components of the sample; Collect at least one fraction of the isolated sample containing the target subcellular components without further centrifugation; and Optionally, the target subcellular components in the sample can be imaged before, during, and / or after separation; The particle size is approximately 1 to approximately 15 micrometers; and wherein: a) The particles form complexes with one or more contaminants, or form structures in the sample medium that interact with one or more contaminants by inhibiting the movement of one or more contaminants in a selected direction relative to the target subcellular component; or b) The particles form a complex with one or more of the pollutants, or form a structure in the sample medium that interacts with one or more pollutants by increasing the movement of one or more pollutants in a selected direction relative to the target subcellular component.

[0014] This document provides a method (“Method 3”) for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells, and cell debris, said method comprising: a) Provide a fluid sample processing apparatus, the fluid sample processing apparatus comprising, (i) Processing channels; (ii) Entrance passage; (iii) An inlet connection area that connects the inlet channel to the processing channel; (iv) Multiple magnetic components aligned along the X-axis of the processing channel on the upper and lower sides of the processing channel; (v) Multiple exit channels; (vi) Export connection area, which connects the processing channel to the export channel; (vii) A first outlet channel, which is in fluid communication with the upper region of the processing channel at the outlet connection area; (viii) A second outlet channel, which is in fluid communication with the lower region of the processing channel at the outlet connection area; and (ix) a first flow regulator associated with the first outlet channel and a second flow regulator associated with the second outlet channel; and b) Flowing the mixture through the fluid sample processing device to provide a first recovered sample rich in the cell nuclei and / or live cells; and a second recovered sample with the cell nuclei and / or live cells removed; The mixture comprises: i) paramagnetic compounds or ferrofluids; and ii) Separate particles (or microbeads) The particle size is approximately 1 to approximately 15 micrometers; and wherein: a) The particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, in a manner that inhibits the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus; or b) The particles form complexes with cell debris, or form structures in the sample medium that interact with cell debris by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

[0015] This document provides a method (“Method 4”) for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells, and nuclear debris, said method comprising: A flowcell cartridge is provided, the flowcell cartridge including a processing channel and a plurality of exit channels, wherein the exit channels of the flowcell cartridge have a volume larger than that of the processing channel; The sample solution containing live and dead cells, as well as paramagnetic compounds, flows into the processing channel; The flow chamber cartridge is placed in a magnetic field that is substantially parallel to and aligned with the processing channel; The processing channel and the sample contained therein are held completely within the magnetic field in a stopped-flow state for a sufficient time to allow live and dead cells to separate vertically within the processing channel; and Simultaneously, sample fractions rich in live cells and / or cell nuclei and sample fractions rich in dead cells and cell nucleus debris are discharged into the outlet channel; The sample solution contains separated particles with a size of about 1 to about 15 micrometers; and wherein: a) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or b) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

[0016] This article provides a kit (“Kit 1”) for isolating cell nuclei, which contains one or more components selected from buffers, solvents, proteins, particles, enzymes, stabilizers and the like.

[0017] This document provides any of the aforementioned methods 2, 3, or 4 performed on a fluid concentrator apparatus, the fluid concentrator apparatus comprising an inlet channel, a processing channel, and at least two outlet channels, and a pump for moving a particulate sample through the concentrator apparatus. The concentrator apparatus may have a separate diversion channel, which may be controlled by a valve or functionally similar diversion technology to collect all or part of the concentrated or depleted particulate stream. The concentrator apparatus may operate under automatic control and also includes one or more sensors located within or near a portion of the processing or inlet channel to detect the presence or quantity of particles, or other physical or chemical properties of the particulate or sample flow. The detector output may be operatively connected to a concentrator controller to optimize concentration and stratification processing conditions. Particle concentration / removal may be physically achieved by the apparatus via gravity settling, magnetic levitation / repulsion, or combinations thereof. The interface between the inlet channel and the processing channel is preferably geometrically configured to reduce or eliminate turbulence in the processing channel. The interface between the processing channel and the outlet channel is preferably geometrically oriented to facilitate the collection of the stratified stream, the particle enrichment stream, and the particle depletion stream into their respective outlet channels.

[0018] This document also provides any of the aforementioned methods 2, 3, or 4, performed on a fluid concentrator having a magnetic assembly positioned substantially linearly along a processing channel (X-axis) to provide magnetic repulsion or attraction of particles in the processing channel based on the paramagnetic properties of the particles. The magnetic assembly can induce or enhance particle settling within the processing channel. Another application of the fluid concentrator, which includes a magnetic assembly along the processing channel, is that it can operate in low-gravity or microgravity environments. Alternatively, in some cases, when the sample fluid is a mixture of heterogeneous particles, the magnetic assembly can selectively suppress the settling of certain particles in the sample fluid. In some embodiments, providing a magnetic field within the inlet channel can produce a pre-concentration effect by providing an overcoming impediment to the flow of particles flowing from the inlet channel into the processing channel. An inlet channel magnetic field can be induced by extending magnets arranged substantially linearly along the processing channel into the inlet channel. Alternatively, the magnetic assembly can be placed independently of the processing channel and magnetically connected to the inlet channel. In one embodiment, this is a bar magnet; in another embodiment, it is a ring or annular magnet surrounding all or part of the inlet channel. The inlet channel magnet can be a permanent magnet or an electromagnet controlled by a magnetic controller. In another embodiment illustrated below, the processing channel can have multiple magnetic components arranged substantially linearly with respect to the processing channel. In one such embodiment, magnets providing different magnetic field strengths are positioned relative to each other (e.g., top and bottom) in a substantially linear manner parallel to the processing channel. When combined with a pre-concentration step or apparatus configuration, this embodiment can selectively function to concentrate a predetermined particulate component of a sample containing a heterogeneous particulate composition. Particle accumulation outside the processing channel can be a passive process, depending on the sample flow rate and the mobility of the particles in the sample liquid medium within the inlet channel. Particle accumulation outside the processing channel can be an active process, utilizing the magnetic field within the inlet channel to impede the particles within the field. The impediment to particle movement from the inlet channel into the processing channel can be overcome by controlling the flow rate or flow pattern. For example, particle mobility in the inlet channel can be increased by increasing the flow rate or introducing one or more pulses that increase the channel pressure. When particle suppression is achieved by inducing an electromagnetic field in the inlet channel, the mobility of particles entering the processing channel from the inlet channel can be increased by reducing the magnetic field in the inlet channel, changing the flow rate or pressure in the inlet channel, or a combination of magnetic field and sample flow properties.

[0019] This document also provides any of the aforementioned methods 2, 3, or 4 performed on a particle concentrator comprising a fluid processing channel structure, at least one magnetic component, and at least two output ports, wherein the fluid processing channel comprises a substantially linear portion having a front end in fluid communication with the input port and a tail end in fluid communication with the output port. The at least two output ports are configured substantially in parallel. According to this embodiment, each output port includes at least one collection path leading to a collection chamber containing a defined amount of material required for subsequent processing steps. The fluid channel structure is typically a microcapillary channel, allowing particles to flow freely or at a desired rate. The device may also include one or more pumps configured to drive fluid from the input port through the fluid channel structure. In some embodiments, the device further includes one or more valves for controlling the particle path and / or flow rate.

[0020] Embodiments of the method of the present invention are further described in the following numbered embodiments. These numbered embodiments are not intended to limit the invention and may be combined with other elements and alternatives described herein.

[0021] Implementation plan of method 1

[0022] In a first embodiment, this document provides a method (“Method 1”) for extracting cell nuclei from intact cells, comprising: providing intact cells; and lysing the cells in a nucleus separation buffer; wherein the nucleus separation buffer contains wheat germ lectin (WGA).

[0023] In the second embodiment (2), the WGA content is from about 0.01 mg / mL to about 2 mg / mL; for example, from about 0.01 mg / mL to about 1 mg / mL; for example, from about 0.01 mg / mL to about 0.5 mg / mL; for example, from 0.05 mg / mL to about 0.15 mg / mL; for example, about 0.1 mg / mL. In the third embodiment (3), the cell nucleus separation buffer comprises the WGA and one or more of the following: buffer, solvent, protein, particles, enzyme, stabilizer and solvent; and water.

[0024] In the fourth embodiment (4), the method further includes, for example, collecting cell nuclei by centrifugation; and optionally storing the cell nuclei; for example, by suspending the cell nuclei in a storage buffer; wherein the storage buffer contains, for example, sucrose. In the fifth embodiment (5), the integrity of the isolated cell nuclei is at least about 15% higher than that of cell nuclei isolated by a similar method lacking WGA. In the sixth embodiment (6), the method further includes suspending the collected cell nuclei in a suspension buffer containing a suspending agent. In the seventh embodiment (7), the suspending agent contains 100 mM gadolinium, and the suspension buffer contains 1×PBS, 1% BSA, and an RNAse inhibitor, such as RNAseOUT. TM In the eighth embodiment (8), the method further includes using the isolated cell nucleus to perform any of the methods 2 and subsequent methods described herein.

[0025] In another embodiment, this disclosure provides a method (“Method 1a”) for reducing nuclear contamination from environmental RNA, comprising isolating cell nuclei in the presence of wheat germ lectin (WGA). In the second embodiment (2), the WGA concentration is from about 0.01 mg / mL to about 2 mg / mL; for example, from about 0.01 mg / mL to about 1 mg / mL; for example, from about 0.01 mg / mL to about 0.5 mg / mL; for example, from 0.05 mg / mL to about 0.15 mg / mL; for example, from about 0.1 mg / mL.

[0026] Implementation plan of method 2

[0027] This article provides a method (“Method 2”) for separating target subcellular components, such as cell nuclei, from a sample containing target subcellular components and one or more contaminants, comprising: A sample comprising the target subcellular component, the contaminant, and the sample medium is loaded into a separation channel, wherein the sample optionally flows along the separation channel, and wherein the sample medium comprises: i) paramagnetic compounds or ferrofluids; and ii) Separate particles or microbeads; At least one magnet is used to apply a magnetic force to the sample in order to separate the target subcellular component from other components of the sample; Collect at least one fraction of the isolated sample containing the target subcellular components without further centrifugation; and Optionally, the target subcellular components in the sample can be imaged before, during, and / or after separation; The particle size is approximately 1 to approximately 15 micrometers; and wherein: a) Separating particles from one or more contaminants to form complexes, or to form structures in the sample medium that interact with one or more contaminants, by means of inhibiting the movement of one or more contaminants in a selected direction relative to the target subcellular component in the same direction; or b) The separated particles form complexes with the one or more contaminants, or form structures in the sample medium that interact with the one or more contaminants, by means of increasing the movement of the one or more contaminants in a selected direction relative to the target subcellular components moving in the same direction.

[0028] In the second embodiment (2) of method 2, the separated particles form a complex with one or more pollutants, or form a structure in the sample medium that interacts with one or more pollutants, in a way that inhibits the movement of one or more pollutants relative to the target subcellular components in the direction of environmental gravity.

[0029] The third embodiment (3) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the target subcellular component is the cell nucleus. The fourth embodiment (4) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the contaminant includes one or more of intact cells, dead cells, nuclear fragments, and cell fragments. The fifth embodiment (5) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the size of the separated particles is about 1 to about 10 micrometers; for example, about 1 to about 8 micrometers; for example, about 1 to about 5 micrometers; for example, about 1, about 2, about 3, about 4, or about 5 micrometers. The sixth embodiment (6) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the separated particles are selected from agarose microbeads such as Sepharose. TM Microbeads, dextran microbeads such as Sephadex TM Polyacrylamide microbeads, glucose microbeads, polystyrene microbeads, microbeads made of polymer resins such as polyvinylethyl carbitol, polyvinylpyrrolidone, cellulose, silicon-based materials, and / or microbeads made of mixtures such as dextran-polyacrylamide such as Sephacryl™ microbeads; each of these microbeads may optionally be coated, for example, with proteins.

[0030] The seventh embodiment (7) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the separated particles are optionally coated polystyrene particles; for example, polystyrene particles coated with protein. The eighth embodiment (8) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the separated particles are polystyrene particles coated with streptavidin. The ninth embodiment (9) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the size of the separated particles is about 3 micrometers. The tenth embodiment (10) of Method 2 is an embodiment of any of the preceding embodiments of Method 2, wherein the sample contains about 50 to about 10,000,000 cell nuclei.

[0031] The eleventh embodiment (11) of Method 2 is an embodiment of any of the foregoing embodiments of Method 2, wherein the concentration of cell nuclei in the fraction is increased by at least 1.1:1 compared to the original sample. The twelfth embodiment (12) of Method 2 is an embodiment of any of the foregoing embodiments of Method 2, wherein the concentration of non-nuclear particles in the original sample is reduced by at least about 1% in the fraction. The thirteenth embodiment (13) of Method 2 is an embodiment of any of the foregoing embodiments of Method 2, wherein the integrity of the isolated cell nuclei from the sample fraction is at least about 30% higher than the integrity of cell nuclei isolated from the sample fraction by methods including centrifugation.

[0032] The fourteenth embodiment (14) of Method 2 is an embodiment of any of the foregoing embodiments of Method 2, wherein the cell nucleus is isolated from human cells, non-human animal cells, or plant cells. The fifteenth embodiment (15) of Method 2 is an embodiment of any of the foregoing embodiments of Method 2, wherein the cell nucleus is isolated from healthy cells, diseased cells, infected cells, transfected cells, or genetically modified cells.

[0033] Implementation plan of method 3

[0034] This document provides a method (“Method 3”) for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells, and cell debris, said method comprising: A) Provide a fluid sample processing apparatus, the fluid sample processing apparatus comprising: (i) Processing channels; (ii) Entrance passage; (iii) An inlet connection area that connects the inlet channel to the processing channel; (iv) Multiple magnetic components aligned along the X-axis of the processing channel on the upper and lower sides of the processing channel; (v) Multiple exit channels; (vi) Export connection area, which connects the processing channel to the export channel; (vii) A first outlet channel, which is in fluid communication with the upper region of the processing channel at the outlet connection area; (viii) A second outlet channel, which is in fluid communication with the lower region of the processing channel at the outlet connection area; and (ix) a first flow regulator associated with the first outlet channel and a second flow regulator associated with the second outlet channel; and B) Flow the mixture through the fluid sample processing device to provide a first recovered sample rich in the cell nuclei and / or live cells; and a second recovered sample with the cell nuclei and / or live cells removed; The mixture comprises: i) paramagnetic compounds or ferrofluids; and ii) Separate particles; The size of the separated particles is approximately 1 to approximately 15 micrometers; and wherein: a) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or b) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

[0035] The second embodiment (2) of Method 3 is an embodiment in which the separated particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or the third embodiment (3) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the first recovered sample is rich in cell nuclei. The fourth embodiment (4) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the first recovered sample is rich in live cells. The fifth embodiment (5) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the size of the separated particles is about 1 to about 10 micrometers; for example, about 1 to about 8 micrometers; for example, about 1 to about 5 micrometers; for example, about 1, about 2, about 3, about 4 or about 5 micrometers.

[0036] The sixth embodiment (6) of method 3 is an embodiment of any of the foregoing embodiments of method 3, wherein the separated particles are selected from agarose microbeads such as Sepharose. TM Microbeads, dextran microbeads such as Sephadex TMThe microbeads include polyacrylamide microbeads, glucose microbeads, polystyrene microbeads, microbeads made from polymeric resins such as polyvinylethyl carbitol, polyvinylpyrrolidone, cellulose, and silicon-based materials, and / or microbeads made from mixtures such as dextran-polyacrylamide, such as Sephacryl™ microbeads; each type of microbead may optionally be coated, for example, with a protein. A seventh embodiment (7) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the separating particles are optionally coated polystyrene particles; for example, polystyrene particles coated with a protein. An eighth embodiment of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the separating particles are polystyrene particles coated with streptavidin.

[0037] The ninth embodiment (9) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the size of the separated particles is about 3 micrometers. The tenth embodiment (10) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the sample contains about 50 to about 10,000,000 cell nuclei. The eleventh embodiment (11) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the concentration of cell nuclei in the fraction is increased by at least 1.1:1 compared to the original sample. The twelfth embodiment (12) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the concentration of non-nuclear particles in the original sample is reduced by at least about 1% in the fraction. The thirteenth embodiment (13) of Method 30 is an embodiment of any of the preceding embodiments of Method 3, wherein the integrity of the separated cell nuclei from the sample fraction is at least about 30% higher than the integrity of cell nuclei separated from the sample fraction by methods including centrifugation.

[0038] The fourteenth embodiment (14) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the cell nuclei are isolated from human cells, non-human animal cells, or plant cells. The fifteenth embodiment (15) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the cell nuclei are isolated from healthy cells, diseased cells, infected cells, transfected cells, or genetically modified cells. The sixteenth embodiment (16) of Method 3 is an embodiment of any of the preceding embodiments of Method 3, wherein the yield of live cells in the first recovered sample is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the mixture; and / or the yield of cell nuclei in the first recovered sample is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total cell nuclei from the live cell composition of the mixture.

[0039] The seventeenth embodiment (17) of method 3 is an embodiment of any of the foregoing embodiments of method 3, wherein the outlet connection area further includes a flow splitter portion. The eighteenth embodiment (18) is an embodiment of the seventeenth embodiment (17) of method 3, wherein the flow splitter portion is advanced into the processing channel and is configured and arranged to separate the fluid flow into separate flows in the outlet channel.

[0040] The nineteenth embodiment (19) of Method 3 is an embodiment of any of the foregoing embodiments of Method 3, wherein the fluid sample processing apparatus further includes a first flow rate sensor associated with a first outlet channel and a second flow rate sensor associated with a second outlet channel. The twentieth embodiment (20) of Method 3 is an embodiment of embodiment 19, wherein the flow rate sensor is operatively linked to a flow regulator.

[0041] Implementation scheme twenty-one (21) is an implementation scheme of any of the foregoing implementation schemes of method 3, wherein the fluid sample processing apparatus further includes an optical sensor and an illumination source, the illumination source being configured to be opposite to or angularly adjacent to the optical sensor; optionally wherein the illumination source emits ultraviolet light.

[0042] Implementation scheme twenty-two (22) is an implementation scheme according to any of the foregoing implementation schemes of method 3, wherein the fluid sample processing apparatus further includes a sensor, wherein the sensor is a photodetector, a multi-pixel imaging detector, a magnetic field detector, an electrochemical detector, an optical phase detector, a scattering detector, a Hall sensor, a magnetoresistive sensor, a radiation thermal sensor, a surface acoustic wave sensor, a biosensor, a capacitive sensor, a conductive sensor, a thermal sensor, a flow rate sensor, an ultrasonic sensor, a gravity sensor, a magnetic field sensor, or a combination thereof; and a controller operatively connected to a plurality of flow regulators.

[0043] Implementation scheme twenty-three (23) is an implementation scheme according to any of the foregoing implementation schemes of method 3, wherein the fluid sample processing apparatus includes a flow chamber cartridge containing a planar substrate, the planar substrate comprising: (i) Upper and lower surfaces; (ii) Forming the first longitudinal side surface of the imaging surface; (iii) Forming a second longitudinal side surface of the illumination surface; as well as (iv) First transverse side and second transverse side; (v) The inlet hole located on the upper surface; (vi) Entrance passage; (vii) Sample processing channel, which is in fluid communication with the inlet channel and is positioned substantially parallel to the longitudinal side; (viii) Sample splitter, located within the processing channel; (ix) Multiple outlet channels, which are in fluid communication with the processing channels; and (x) Multiple collection holes, which are in fluid communication with each of the multiple outlet channels; The substrate may optionally include an optically transparent material, and the processing channel is offset in the plane of the substrate to spatially deflect towards the imaging surface. Optionally, the substrate comprises a non-ferrous metal, ceramic, glass, polymer, or plastic; and Optionally, if the substrate comprises one or more layers, the substrate and the planar layer may comprise the same or different materials.

[0044] Implementation scheme twenty-four (24) is an implementation scheme of any of the foregoing implementation schemes of method 3, wherein: The fluid sample processing apparatus includes a flow chamber cartridge containing a planar substrate, the planar substrate comprising: (i) An inlet hole located on the upper surface; (ii) Entrance passage; (iii) Sample processing channel; (iv) Sample splitter, located within the processing channel; (v) Multiple outlet channels fluidly connected to the processing channels; and (vi) Multiple collection holes, which are in fluid communication with each of the multiple outlet channels; The substrate contains an optically transparent material, and the combined volume of each of the multiple outlet channels is greater than the volume of the processing channel. Optionally, the substrate comprises a non-ferrous metal, ceramic, glass, polymer, or plastic; and Optionally, if the substrate comprises one or more layers, the substrate and the planar layer may comprise the same or different materials.

[0045] Embodiment 25 (25) is an embodiment of any of the foregoing embodiments of Method 3, wherein the outlet channels of the flow chamber cartridge are arranged in a compact path, for example, wherein the outlet channels are serpentine channels. Embodiment 26 (26) is an embodiment of embodiments 23 to 25 of Method 3, wherein the outlet channels of the flow chamber cartridge are formed as recesses within the planar substrate, and a first outlet channel includes a recess on the surface of the planar substrate, and a second outlet channel includes a recess on an opposite side of the planar substrate; optionally, the channels are formed by etching, machining, 3D printing, or molding the planar substrate; and / or the flow chamber further includes one or more additional planar layers positioned above the recesses in the planar substrate to form closed channels; optionally, one or more planar layers are attached to the planar substrate by pressing, adhesive bonding (preferably biocompatible adhesives, more preferably silicone or silicone-based adhesives), solvent bonding, ultrasonic welding, thermal bonding, welding, or 3D printing.

[0046] Embodiment 27 (27) is an embodiment according to embodiments 23 to 26 of method 3, wherein the planar substrate comprises a polymeric material, such as a cyclic olefin polymer or a cyclic olefin copolymer; and further comprises: a collection hole formed on the planar substrate and in fluid communication with a terminal portion of an outlet channel; and / or an internal channel inlet at a first hole height and an internal outlet at a second hole height, wherein the inlet is in fluid communication with an outlet channel of a flow chamber cartridge, and wherein the second hole height is higher than the first hole height.

[0047] Implementation plan of method 4

[0048] This document provides a method (“Method 4”) for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells, and nuclear debris, said method comprising: A flow chamber cartridge is provided, the flow chamber cartridge including a processing channel and a plurality of outlet channels, wherein the outlet channels of the flow chamber cartridge have a volume larger than that of the processing channel; Sample solutions containing live cells and / or cell nuclei, dead cells and cell nucleus fragments, as well as paramagnetic compounds, are allowed to flow into the processing channel; The flow chamber cartridge is placed in a magnetic field that is substantially parallel to and aligned with the processing channel; The processing channel and the sample contained therein are held completely within the magnetic field in a stopped-flow state for a sufficient time to allow live and dead cells to separate vertically within the processing channel; and Simultaneously, sample fractions rich in live cells and / or cell nuclei and sample fractions rich in dead cells and / or intact cells and cell nucleus fragments are discharged into the outlet channel; The sample solution and / or flow stream further contain separated particles with a size of about 1 to about 15 micrometers; and wherein: a) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or b) Separating particles from cell debris to form complexes, or forming structures in a sample medium that interact with cell debris, by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

[0049] The second embodiment (2) of Method 4 is an embodiment in which the separated particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or the third embodiment (3) of Method 4 is an embodiment according to any of the foregoing embodiments of Method 4, further comprising providing a flow chamber cartridge that is substantially free of any liquid or paramagnetic compound before the introduction of the sample solution. The fourth embodiment (4) of Method 4 is an embodiment according to any of the foregoing embodiments of Method 4, wherein the size of the separated particles is about 1 to about 10 micrometers; for example, about 1 to about 8 micrometers; for example, about 1 to about 5 micrometers; for example, about 1, about 2, about 3, about 4 or about 5 micrometers.

[0050] The fifth embodiment (5) of method 4 is an embodiment of any of the foregoing embodiments of method 4, wherein the separated particles are selected from agarose microbeads such as Sepharose. TM Microbeads, dextran microbeads such as Sephadex TM Polyacrylamide microbeads, glucose microbeads, polystyrene microbeads, microbeads made of polymer resins such as polyvinylethyl carbitol, polyvinylpyrrolidone, cellulose, silicon-based materials, and / or microbeads made of mixtures such as dextran-polyacrylamide such as Sephacryl™ microbeads; each of these microbeads may optionally be coated, for example, with proteins.

[0051] The sixth embodiment (6) of Method 4 is an embodiment according to any of the preceding embodiments of Method 4, wherein the separated particles are optionally coated polystyrene particles; for example, polystyrene particles coated with protein. The seventh embodiment (7) of Method 4 is an embodiment according to any of the preceding embodiments of Method 4, wherein the separated particles are polystyrene particles coated with streptavidin. The eighth embodiment (8) of Method 4 is an embodiment according to any of the preceding embodiments of Method 4, wherein the size of the separated particles is about 3 micrometers. The ninth embodiment (9) of Method 4 is an embodiment according to any of the preceding embodiments of Method 4, wherein the sample contains about 50 to about 10,000,000 cell nuclei. The tenth embodiment (10) of Method 4 is an embodiment according to any of the preceding embodiments of Method 4, wherein the concentration of cell nuclei in the fraction is increased by at least 1.1:1 compared to the original sample.

[0052] The eleventh embodiment (11) of Method 4 is an embodiment of any of the preceding embodiments of Method 4, wherein the concentration of non-nuclear particles in the original sample is reduced by at least about 1% in the fraction. The twelfth embodiment (12) of Method 4 is an embodiment of any of the preceding embodiments of Method 4, wherein the integrity of the isolated nuclei from the sample fraction is at least about 30% higher than the integrity of the nuclei isolated from the sample fraction by means including centrifugation. The thirteenth embodiment (13) of Method 4 is an embodiment of any of the preceding embodiments of Method 4, wherein the nuclei are isolated from human cells, non-human animal cells, or plant cells. The fourteenth embodiment (14) of Method 4 is an embodiment of any of the preceding embodiments of Method 4, wherein the nuclei are isolated from healthy cells, diseased cells, infected cells, transfected cells, or genetically modified cells.

[0053] The fifteenth embodiment (15) of method 4 is an embodiment of any of the foregoing embodiments of method 4, and further includes providing a flow chamber cartridge, wherein the cross-sectional area of ​​the outlet channel is smaller than the cross-sectional area of ​​the processing channel and is arranged in a compact path arrangement, an exemplary configuration being a serpentine channel.

[0054] The sixteenth embodiment (16) of method 4 is an embodiment of any of the foregoing embodiments of method 4, further comprising providing magnetic fields at the top vertical surface immediately adjacent to the processing channel and at the bottom vertical surface immediately adjacent to the processing channel, each magnetic field having similar strength and surface field strength between about 0.8 Tesla and about 2.0 Tesla and optionally between about 0.9 Tesla and about 1.4 Tesla.

[0055] The seventeenth embodiment (17) of method 4 is an embodiment of any of the foregoing embodiments of method 4, further comprising providing a paramagnetic compound in the sample solution at a concentration of about 50 mM to about 200 mM, optionally about 65 mM to about 175 mM, and further optionally about 70 mM to about 150 mM.

[0056] The eighteenth embodiment (19) of method 4 is an embodiment of any of the foregoing embodiments of method 4, further comprising the step of discharging sample fractions into an outlet channel at a flow rate of about 75 uL / min to about 150 uL / min, and optionally about 75 uL / min, about 90 uL / min, about 100 uL / min, about 110 uL / min, about 120 uL / min or about 150 uL / min.

[0057] The nineteenth embodiment (19) of Method 4 is an embodiment of any of the preceding embodiments of Method 4, wherein the enriched recovered sample fraction contains at least about 60%, at least about 70%, at least about 80%, or at least about 90% live cells.

[0058] The twentieth embodiment (20) of Method 4 is an embodiment of any of the foregoing embodiments of Method 4, wherein the yield of live cells in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample.

[0059] The twenty-first embodiment (21) of Method 4 is an embodiment of any of the foregoing embodiments of Method 4, wherein the yield of cell nuclei in the enriched recovered sample fraction is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total cell nuclei composed of live cells from the sample.

[0060] Other methods and implementation schemes

[0061] This document provides a method (method 5) for concentrating cell and / or nucleus samples, comprising (i) providing a low-volume fluid device having a processing channel, an inlet channel, and multiple outlet channels; (ii) under certain conditions, allowing a sample containing cells and / or nuclei to flow into the processing channel through the inlet channel to generate a sample flow having at least a cell and / or nucleus enrichment layer and a cell and / or nucleus removal layer; (iii) allowing the cell and / or nucleus enrichment layer to flow through a first outlet channel to generate a cell and / or nucleus enriched flow; (iv) allowing the cell and / or nucleus removal layer to flow through a second outlet channel to generate a cell and / or nucleus removed flow; and (v) collecting one or more flow streams from one or more outlet channels; wherein the sample and / or flow stream comprises separated particles with a size of about 1 to about 15 micrometers; and wherein: a) The separated particles form complexes with one or more non-nuclear components in the sample, or form structures in the flow stream that interact with one or more non-nuclear components in the sample by inhibiting the movement of one or more non-nuclear components in the sample in a selected direction relative to the movement of the nucleus; or b) The separated particles form a complex with one or more non-nuclear components in the sample, or form a structure in the sample medium that interacts with one or more non-nuclear components in the sample by increasing the movement of one or more non-nuclear components in the sample in a selected direction relative to the movement of the nucleus.

[0062] The second embodiment (2) of Method 5 is the first embodiment, which further includes subjecting the sample containing cells and / or nuclei to flow conditions sufficient to induce the sedimentation of sample cells and / or nuclei into a flow stream enriched with cells and / or nuclei. The third embodiment (3) of Method 5 is the first embodiment (1), which further includes providing a magnetic field from the top of the processing channel and aligned with the X-axis of the processing channel, and repelling cells and / or nuclei in the sample into the flow stream enriched with cells and / or nuclei. The fourth embodiment (4) of method 5 is embodiment three or four, which further includes: (i) inducing a magnetic field in the inlet channel to impede the movement of cells and / or nuclei from the inlet channel into the processing channel to form a concentrated portion of the sample flow in the inlet channel; (ii) moving the concentrated portion of the sample flow into the processing channel; (iii) generating a flow enriched with cells and / or nuclei; (iv) allowing the flow enriched with cells and / or nuclei to flow through the outlet channel; and (v) capturing the flow enriched with cells and / or nuclei.

[0063] The fifth embodiment (5) of method 5 is a method of embodiment two or three (2-3), which further includes: (i) inducing a magnetic field in the inlet channel to impede the movement of cells and / or nuclei from the inlet channel into the processing channel to form a cell and / or nucleus concentration section of the sample flow in the inlet channel; (ii) moving an unimpeded portion of the sample flow into the processing channel; (iii) generating a cell and / or nucleus removal flow; (iv) allowing the cell and / or nucleus removal flow to flow through the outlet channel; and (v) capturing the cell and / or nucleus removal flow.

[0064] The sixth embodiment (6) of Method 5 is an embodiment of 1 to 4 (1-4), further comprising: measuring cells and / or nuclei in the cell and / or nucleus enrichment layer in the processing channel to determine relative cell and / or nucleus concentration or location, and collecting fractions of the cell and / or nucleus enrichment layer based on high relative cell and / or nucleus concentration or location in the processing channel. The seventh embodiment (7) of Method 5 is an embodiment of 1 to 3 (1-3) or embodiment 5 (5), further comprising: measuring cells and / or nuclei in the cell and / or nucleus removal layer in the processing channel to determine relative cell and / or nucleus concentration, and collecting fractions of the cell and / or nucleus removal layer based on low relative cell and / or nucleus concentration.

[0065] The eighth embodiment (8) of Method 5 is Embodiment 5 (5), which further includes providing a magnetic field within the inlet channel that is continuous with the magnetic field generated from the top of the processing channel and aligned along the X-axis of the processing channel. The ninth embodiment (9) of Method 5 is Embodiment 5 (5), which further includes generating a ring-shaped magnetic field around the inlet channel. The tenth embodiment (10) of Method 5 is the method of Embodiment 9 (9), which further includes modulating the magnetic field within the inlet channel to facilitate the movement of cell and / or nuclear enrichment segments within the inlet channel into the processing channel.

[0066] The eleventh embodiment (11) of Method 5 is the method of Embodiments 1 to 10 (1-10), which further includes detecting the properties of cells and / or nuclei within the processing channel and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The twelfth embodiment (12) of Method 5 is the method of Embodiment 11 (11), which further includes detecting the properties of cells and / or nuclei within the cell and / or nucleus-enriched flow stream and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The thirteenth embodiment (13) of Method 5 is the method of Embodiments 10 or 12 (10 or 12), which further includes detecting the properties of cells and / or nuclei within the processing channel and adjusting the magnetic field within the inlet channel to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream.

[0067] The fourteenth embodiment (14) of method 5 is the method of embodiment thirteen (13), which further includes detecting the properties of cells and / or nuclei in the flow stream enriched with cells and / or nuclei, and modulating the magnetic field in the inlet channel to manipulate the concentration of cells and / or nuclei in the flow stream enriched with cells and / or nuclei.

[0068] The fifteenth embodiment (15) of Method 5 is the method of Embodiments 1 to 10 (1-10), which further includes detecting the cellular and / or nuclear properties within the inlet channel and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The sixteenth embodiment (16) of Method 5 is the method of Embodiment 10 (10), which further includes detecting the cellular and / or nuclear properties within the inlet channel and adjusting the magnetic field within the inlet channel to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The seventeenth embodiment (17) of Method 5 is the method according to Embodiments 11 to 16 (11-16), which further includes detecting the relative cell and / or nucleus concentration or cell and / or nucleus density. The eighteenth embodiment (18) of Method 5 is the method according to Embodiments 10 to 17 (10-17), which further includes detecting the chemical properties within the processing channel and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The nineteenth embodiment (19) of method 5 is the method according to embodiments 10 to 17 (10-17), which further includes detecting the chemical properties within the flow stream enriched with cells and / or nuclei, and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the flow stream enriched with cells and / or nuclei.

[0069] The twentieth embodiment (20) of Method 5 is the method according to Embodiments 10 to 17 (10-17), which further includes detecting the chemical properties within the processing channel and adjusting the magnetic field within the inlet channel to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The twenty-first embodiment (21) of Method 5 is the method according to Embodiment 20 (20), which further includes detecting the chemical properties within the cell and / or nucleus-enriched flow stream and adjusting the magnetic field within the inlet channel to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream. The twenty-second embodiment (22) of Method 5 is the method according to Embodiments 1 to 21 (1-21), which further includes detecting the chemical properties within the inlet channel and adjusting the sample flow rate to manipulate the concentration of cells and / or nuclei within the cell and / or nucleus-enriched flow stream.

[0070] Implementation scheme 23 (23) of method 5 is based on the method of implementation schemes 10 to 22 (10-22), which further includes detecting the chemical properties within the inlet channel and adjusting the magnetic field within the inlet channel to manipulate the concentration of cells and / or nuclei in the flow stream enriched with cells and / or nuclei.

[0071] Embodiment 24 (24) of Method 5 is the method of Embodiments 18 to 23 (18-23), wherein the property is an electrochemical, photonic, spectroscopic, or binding property. Embodiment 25 (25) of Method 5 is the method according to Embodiments 1 to 24 (1-24), which further includes diverting the flow stream of cell and / or nucleus removal into a collection channel and capturing fractions of the flow stream of cell and / or nucleus removal.

[0072] Implementation scheme 26 (26) of method 5 is a method according to implementation schemes 1 to 25 (1-25), which further includes diverting the flow stream enriched with cells and / or nuclei into a collection channel and capturing fractions of the flow stream enriched with cells and / or nuclei.

[0073] Implementation scheme twenty-seven (27) of method 5 is the method according to implementation schemes one through twenty-six (1-26), which further includes redirecting the flow stream for cell and / or nucleus removal and redirecting the flow stream for cell and / or nucleus enrichment to the corresponding collection channel, and capturing fractions of each flow stream. Implementation scheme twenty-eight (28) of method 5 is the method according to implementation scheme twenty-seven (27), which further includes capturing multiple discrete fractions of the flow streams. Implementation scheme twenty-nine (29) of method 5 is the method according to implementation scheme twenty-seven or twenty-eight (27 or 28), which further includes capturing non-simultaneous fractions from the flow streams for cell and / or nucleus removal and the flow streams for cell and / or nucleus enrichment.

[0074] Method 5, embodiment 30 (30), is based on embodiments 1 to 29 (1-29), further comprising adding a paramagnetic compound to the sample prior to introduction into the inlet channel. Method 5, embodiment 31 (31), is based on embodiments 1 to 30 (1-30), further comprising performing subsequent reactions on the separated fractions. Method 5, embodiment 32 (32), is based on embodiment 31 (31), wherein the subsequent reactions are binding, PCR, sequencing sample preparation, enzymatic degradation, or enzymatic synthesis reactions. Method 5, embodiment 33 (33), is based on embodiment 31 (31), wherein the collected samples are subjected to cell culture, fluorescence-activated cell sorting, or magnetic levitation cell sorting.

[0075] Implementation of Method 5, 34 (34), is a method according to any one of Implementations 1 to 33 (1-33), wherein the sample fluid first flows at an angle that is substantially not linearly aligned with the processing channel, and then flows at an angle that is substantially linear with the processing channel.

[0076] Method 5, embodiment thirty-five (35), is a method for grading blood samples, comprising (i) providing a whole blood sample or a diluted blood sample, and (ii) subjecting the sample to the sample concentration methods of embodiments one through thirty-four (1-34), and separating plasma and / or blood cells from the whole blood sample or the diluted blood sample. Method 5, embodiment thirty-six (36), is the method of embodiment thirty-five (35), wherein the volume of the blood sample is about 50 μL to about 10 mL. Method 5, embodiment thirty-seven (37), is the method of embodiment thirty-six (36), wherein the plasma fraction contains less than about 1% blood cells in the blood sample. Method 5, embodiment thirty-eight (38), is the method of embodiment thirty-seven (37), wherein the plasma fraction contains less than about 0.01% blood cells in the blood sample. Method 5, embodiment thirty-nine (39), is the method of embodiment thirty-eight (38), wherein the plasma fraction is substantially free of blood cells from the blood sample. The fortieth embodiment (40) of Method 5 is the method according to embodiments 35 to 39 (35-39), wherein the blood sample is a peripheral blood sample, an umbilical cord blood sample, a fetal blood sample, or an arterial blood sample. The forty-first embodiment (41) of Method 5 is the method according to embodiments 35 to 40 (35-40), which further includes a diagnostic assay of the separated fraction. The forty-second embodiment (42) of Method 5 is the method according to embodiment 41 (41), wherein the assay is an enzyme immunoassay, a chemiluminescent immunoassay, a hemagglutination / granule agglutination assay, a nucleic acid amplification assay, a pharmacological assay, a forensic assay, or a genetic trait assay. The forty-third embodiment (43) of Method 5 is an embodiment of the method according to any one of embodiments 1 to 41 (1-41), wherein the reaction is performed on the cells and / or nuclei or the components of the cell and / or nucleus removal layer within the inlet channel and / or processing channel, and optionally simultaneously with cell and / or nucleus separation / concentration. Implementation scheme 44 (44) of method 5 is an implementation scheme of implementation scheme 43 (43) of method, wherein the reaction is a binding reaction or a staining reaction.

[0077] Implementation scheme 45 (45) of method 5 is an implementation scheme of any one of implementation schemes 1 (1) to 44 (44), wherein the particle is a cell nucleus.

[0078] Reagent Kit Implementation Plan

[0079] This document provides a kit (“Kit 1”) for isolating cell nuclei, comprising a nucleus isolation buffer and one or more additional buffers, solvents, proteins, particles, enzymes and / or stabilizers. A second embodiment (2) of Kit 1 is an embodiment in which the isolation particles comprise microbeads, such as polystyrene microbeads, optionally coated with a protein such as streptavidin, having a size of about 1 micrometer to about 10 micrometers; for example, a size of about 1 to about 8 micrometers; for example, a size of about 1 to about 5 micrometers; for example, a size of about 1, about 2, about 3, about 4 or about 5 micrometers.

[0080] The third embodiment (3) of kit 1 is an embodiment according to any of the preceding embodiments of kit 1, wherein the separated particles comprise microbeads with a size of about 1 to about 8 micrometers; for example, with a size of about 1 to about 5 micrometers; for example, with a size of about 1, about 2, about 3, about 4 or about 5 micrometers. The fourth embodiment (4) of kit 1 is an embodiment according to any of the preceding embodiments of kit 1, wherein the size of the separated particles is about 3 micrometers.

[0081] The fifth embodiment (5) of kit 1 is an embodiment according to any of the foregoing embodiments of kit 1, wherein one or more components of the kit are provided in a solid form for easy reconfiguration. The sixth embodiment (6) of kit 1 is an embodiment according to any of the foregoing embodiments of kit 1, further comprising a gadolinium-containing suspending agent.

[0082] Apparatus Implementation Scheme for Carrying Out the Method of the Invention

[0083] Other embodiments of the apparatus suitable for implementing the methods disclosed herein are further described in the following numbered embodiments. These numbered embodiments are not intended to limit the invention and may be combined with other elements and alternatives described herein.

[0084] The first embodiment (1) is a magnetic fluid sample processing device comprising (i) a processing channel; (ii) an inlet channel; (iii) an inlet connection region connecting the inlet channel to the processing channel; (iv) a plurality of outlet channels; (v) an outlet connection region connecting the processing channel to the outlet channels; (vi) a first outlet channel in fluid communication with an upper region of the processing channel at the outlet connection region; (vii) a second outlet channel in fluid communication with a lower region of the processing channel at the outlet connection region; and (viii) a magnet aligned along the X-axis of the processing channel on the upper and lower sides of the processing channel. The second embodiment (2) is a magnetic fluid sample processing device comprising (i) a processing channel; (ii) an inlet channel; (iii) an inlet connection region connecting the inlet channel to the processing channel; (iv) a plurality of outlet channels; (v) an outlet connection region connecting the processing channel to the outlet channels; (vi) a first outlet channel in fluid communication with an upper region of the processing channel at the outlet connection region; (vii) a second outlet channel in fluid communication with a lower region of the processing channel at the outlet connection region; and (viii) a plurality of magnetic components aligned along the X-axis of the processing channel on the upper and lower sides of the processing channel, wherein the processing device is configured and arranged to pre-concentrate particles before introduction into the processing channel.

[0085] The third embodiment (3) is a fluid sample processing device, comprising (i) a processing channel; (ii) an inlet channel; (iii) an inlet connection region that connects the inlet channel to the processing channel; (iv) a plurality of outlet channels; (v) an outlet connection region that connects the processing channel to the outlet channels; (vi) a first outlet channel that is in fluid communication with an upper region of the processing channel at the outlet connection region; (vii) a second outlet channel that is in fluid communication with a lower region of the processing channel at the outlet connection region; and (viii) an inlet channel flow controller.

[0086] The fourth embodiment (4) is the apparatus according to embodiment 2, wherein the magnetic components located on the upper side and the lower side of the processing channel are constructed and arranged to provide magnetic fields of different intensities within the processing channel.

[0087] The fifth embodiment (5) is the apparatus according to embodiments one through four (1-4), wherein the inlet channel includes a first cross-sectional area and the processing channel includes a second cross-sectional area, wherein the first cross-sectional area is smaller than the second cross-sectional area. The sixth embodiment (6) is the apparatus according to embodiment five (5), wherein the channel is microfluidic or capillary.

[0088] The seventh embodiment (7) is the device according to embodiments one through six (1-6), wherein the inlet connection area tapers gradually at an angle of less than 90 degrees. In the eighth embodiment (8), the device according to embodiment seven (7) is provided, wherein the angle is equal to or less than 60 degrees. In embodiment nine (9), the device according to embodiment eight (8) has a connection angle equal to or less than 45 degrees.

[0089] The tenth embodiment (10) provides an apparatus from embodiments one through nine (1-9), wherein the outlet connection area further includes a flow splitter portion. Embodiment eleven (11) is an apparatus according to embodiment ten (10), wherein the flow splitter portion is advanced into the processing channel and is configured and arranged to separate the respective flow into its outlet channel.

[0090] Implementation scheme 12 (12) is an apparatus according to implementation schemes 1 to 11 (1-11), wherein the first outlet channel includes a first outlet collection channel and a first outlet diversion channel. Implementation scheme 13 (13) provides an apparatus according to implementation scheme 12 (12), wherein the first outlet channel further includes a valve configured and arranged such that the flow in the first outlet channel is in selective fluid communication with either the first outlet collection channel or the first outlet diversion channel.

[0091] Implementation scheme fourteen (14) is an apparatus based on implementation schemes one through thirteen (1-13), wherein the second outlet channel includes a second outlet collection channel and a second outlet diversion channel.

[0092] Embodiment Fifteen (15) is an apparatus according to Embodiment Fourteen (14), wherein the second outlet channel further includes a valve configured and arranged such that the flow in the second outlet channel is in selective fluid communication with a second outlet collection channel or a second outlet diversion channel. Embodiment Sixteen (16) provides an apparatus according to Embodiments One through Fifteen (1-15), wherein the fluid apparatus further includes a magnet that generates a gating magnetic field in the inlet channel or inlet region. Embodiment Seventeen (17) is an apparatus according to Embodiment Sixteen (16), wherein the magnet that generates the gating magnetic field is a ring or annular magnet surrounding the inlet channel or inlet region. In Embodiment Eighteen (18), an apparatus according to Embodiment Sixteen (16) is provided, wherein the magnet that generates the gating magnetic field is aligned with and adjacent to the processing channel and extends into or beyond the channel inlet region of the inlet channel.

[0093] The nineteenth embodiment (19), the apparatus of embodiments one through eighteen (1-18) includes an optically transparent processing channel. In embodiment twenty, the apparatus of embodiments one through eighteen (1-18) includes an optically transparent inlet channel. Embodiment twenty-one provides the apparatus of embodiments one through twenty (1-20), which further includes an inlet channel flow controller, a first outlet channel controller, a second outlet channel controller, or a combination thereof. Embodiment twenty-two (22) is the apparatus according to embodiments B15-B18, which further includes a magnetic field controller (operably linked to a toroidal magnet).

[0094] Embodiment 23 (23) provides the apparatus of Embodiments 1 to 22 (1-22), wherein the fluid apparatus includes one or more sensors. Embodiment 24 (24) is an apparatus according to Embodiment 23 (23), wherein the sensor is selected from optical sensors, capacitive sensors, conductive sensors, thermal sensors, flow rate sensors, ultrasonic sensors, gravity sensors, magnetic field sensors, or combinations thereof. Embodiment 25 (25) is an apparatus according to Embodiment 24 (24), wherein the sensor is a photodetector, multi-pixel imaging detector, magnetic field detector, electrochemical detector, optical phase detector, scattering detector, Hall sensor, magnetoresistive sensor, radiation thermal sensor, surface acoustic wave sensor, biosensor, or combinations thereof.

[0095] The twenty-sixth (26th) embodiment provides an apparatus according to embodiments twenty-three to twenty-five (23-25), wherein the sensor is integrated into or adjacent to the inlet channel. The twenty-seventh embodiment (27th) provides an apparatus according to embodiments twenty-three to twenty-five (23-25), wherein the sensor is integrated into or adjacent to the inlet channel. The twenty-eighth embodiment (28th) provides an apparatus according to embodiments twenty-three to twenty-five (23-25), wherein the sensor is integrated into or adjacent to one or more outlet channels. Embodiment twenty-nine (29th) is an apparatus according to embodiments twenty-three to twenty-five (23-25), wherein the fluid apparatus includes one or more sensors located in or adjacent to the processing channel, one or more sensors located in or adjacent to the inlet channel, one or more sensors located in or adjacent to at least one outlet channel, or a combination thereof.

[0096] Embodiment 30 (30) is an apparatus according to embodiments 25 to 27 (25-27), further comprising an inlet channel flow controller, wherein at least one sensor is operatively connected to the inlet flow controller. Embodiment 31 (31) is an apparatus according to embodiments 25 to 28 (25-28), further comprising an outlet channel flow controller, wherein at least one sensor is operatively connected to the outlet flow controller. Embodiment 32 is an apparatus according to embodiments 23 to 29 (23-29), further comprising an annular or toroidal magnet surrounding an inlet channel or inlet region and a magnetic field controller, wherein a sensor is operatively linked to the magnetic field controller to control the magnetic field of the annular or toroidal magnet.

[0097] Implementation scheme 32 (32) is an apparatus according to implementation schemes 1 to 31 (1-31), wherein the inlet channel further includes a portion substantially linear to the processing channel and a portion substantially nonlinearly aligned to the processing channel, the two portions being connected at a portion at an angle θ, wherein θ ≠ 180° and θ is ≥90°, ≥100°, ≥135°, ≥140°, ≥165° relative to the Y or Z axis or independently relative to the Y and Z axes. 180°, ≥205°, ≥225°, ≥250° or ≤270°.

[0098] Implementation plan for mobile chamber card box

[0099] A first embodiment (1) of the flow chamber cartridge of the present invention includes a planar substrate, the planar substrate including an upper surface and a lower surface, a first longitudinal side surface forming an imaging surface, a second longitudinal side surface forming an illumination surface, a first transverse side surface and a second transverse side surface, an inlet hole located on the upper surface, an inlet channel, a sample processing channel fluidly in communication with the inlet channel and positioned substantially parallel to the longitudinal side surface, a sample splitter located within the processing channel, a plurality of outlet channels fluidly in communication with the processing channel, and a plurality of collection holes fluidly in communication with each of the plurality of outlet channels, wherein the substrate optionally includes an optically transparent material, and wherein the processing channel is offset in the plane of the substrate to be spatially biased toward the imaging surface.

[0100] The second embodiment (2) of the flow chamber cartridge of the present invention includes a planar substrate, the planar substrate including an inlet hole, an inlet channel, a sample processing channel, a sample splitter located in the processing channel, a plurality of outlet channels in fluid communication with the processing channel, and a plurality of collection holes in fluid communication with each of the plurality of outlet channels, wherein the substrate includes an optically transparent material, and wherein the combined volume of each of the plurality of outlet channels is greater than the volume of the processing channel.

[0101] The third implementation scheme (3) is a flow chamber card according to implementation schemes 1 and 2, wherein the outlet channels are arranged in a compact path, and an exemplary configuration is a serpentine channel.

[0102] The fourth embodiment (4) is a flow chamber cartridge according to embodiments 1-3, wherein the outlet channel of the flow chamber cartridge is formed as a recess in a planar substrate, and the first outlet channel includes a recess on the surface of the planar substrate, and the second outlet channel includes a recess on the opposite side of the planar substrate. In embodiments 1-4, the channels are formed by etching, machining, 3D printing, or molding the planar substrate.

[0103] The fifth embodiment (5) is the flow chamber cartridge of embodiment 4, which includes one or more additional planar layers positioned above a recess in a planar substrate to form a closed channel.

[0104] The sixth embodiment (6) includes the flow chamber cartridge of embodiments 1-5, wherein the substrate is made of non-ferrous metal, ceramic, glass, polymer or plastic, and in embodiments having a substrate and one or more layers, the substrate and planar layer may be made of the same or different materials.

[0105] The seventh embodiment (7) of the flow chamber cartridge includes embodiments 5-6, wherein one or more planar layers are attached to a planar substrate by pressing, adhesive bonding, preferably a biocompatible adhesive, more preferably a silicone or silicone-based adhesive, solvent bonding, ultrasonic welding, thermal bonding, welding or 3D printing.

[0106] The eighth embodiment (8) of the flow chamber cartridge includes embodiments 5-7, wherein the planar substrate and one or more planar layers are made of the same material.

[0107] The ninth embodiment (9) of the flow chamber cartridge includes the flow chamber cartridges of embodiments 1-8, wherein the planar substrate comprises a polymer material.

[0108] The tenth embodiment (10) of the flow chamber cartridge includes the flow chamber cartridge of embodiment 9, wherein the polymer material of embodiment 8 includes a cyclic olefin polymer or a cyclic olefin copolymer.

[0109] The eleventh flow chamber cartridge embodiment (11) includes embodiments 1-10, and further includes a collection hole formed on a planar substrate and in fluid communication with the end portion of the outlet channel.

[0110] A twelfth embodiment (12) of the flow chamber cartridge of embodiments 1-11, wherein the collection hole further includes an internal channel inlet at a first hole height and an internal outlet at a second hole height, wherein the inlet is in fluid communication with the outlet channel of the flow chamber cartridge, and wherein the second hole height is higher than the first hole height.

[0111] The thirteenth embodiment (13) of the flow chamber card box includes the flow chamber card box of embodiments 11-12, wherein the collection hole further includes a step that provides an angled transition from the terminal opening of the inlet to the bottom plate of the collection hole.

[0112] The fourteenth embodiment (14) of the flow chamber cartridge includes flow chamber cartridge embodiments 11-13, which also includes a sealing membrane covering the top of one or more collection holes.

[0113] The fifteenth embodiment (15) of the flow chamber cartridge includes flow chamber cartridge embodiments 11-14, wherein the flow chamber cartridge further includes a collection hole outlet channel in fluid communication with a collection hole in a planar substrate.

[0114] Cell or nucleus separation system implementation plan

[0115] The first embodiment (1) of the cell separation system of the present invention includes a receiving block for holding a flow chamber cartridge, an optical system including an optical sensor, a lens and an illumination source, and a plurality of flow adjustment components, wherein the receiving block removably positions the flow chamber cartridge in optical alignment with the optical system, removably engages a magnetic component adjacent to the processing channel of the flow chamber, and removably positions a plurality of outlet channels of the flow chamber cartridge in fluid communication with the plurality of flow adjustment components.

[0116] In the second embodiment (2), embodiment 1 also includes a visible light illumination source configured and arranged to provide light transmission through a processing channel within the planar substrate.

[0117] In the third embodiment (3), the system of embodiments 1-2 further includes one or more ultraviolet illumination sources configured and arranged to orient the ultraviolet illumination (optionally with wavelengths of about 474 nm and / or 560 nm) at an angle relative to the processing channel within the planar substrate held in the receiving block.

[0118] In a fourth embodiment (4) of the cell separation system including embodiment 3, the optical system includes a dual bandpass filter, which preferably allows emitted radiation in a band centered at wavelengths of about 524 nm and 628 nm to pass through. Attached Figure Description

[0119] Figure 1 This is a cross-sectional view of an embodiment of a single magnetic component particle concentration and separation device as described herein.

[0120] Figure 2 This is a cross-sectional view of an embodiment of a single magnetic component particle concentration and separation device with an angled inlet channel as described herein.

[0121] Figure 3 This is a cross-sectional view of an embodiment of a single magnetic component particle concentration and separation device having an angled inlet channel and an inlet magnetic field assembly as described herein.

[0122] Figure 4 This is a cross-sectional view of an embodiment of the particle concentration and separation apparatus as described herein, wherein the magnetic components surround a portion of the inlet channel.

[0123] Figure 5This is a cross-sectional view of an embodiment of a single magnetic component particle concentration and separation device having an angled inlet channel and an inlet magnetic field assembly as described herein.

[0124] Figure 6 A magnetic field applied within a processing channel in a single magnetic component configuration according to the embodiment described herein is depicted.

[0125] Figure 7 This is a cross-sectional view of an embodiment of a multi-magnetic component particle concentration and separation device having angled inlet channels and inlet magnetic field components as described herein.

[0126] Figure 8 This is a cross-sectional view of an embodiment of a particle concentration and separation apparatus having an angled inlet channel and an inlet magnetic field assembly as described herein.

[0127] Figure 9 is a cross-sectional view of an embodiment of the particle concentration and separation device with optional valves and inlet channel pump assembly as described above.

[0128] Figure 10 This is a diagram of the integrated system of the various implementation schemes described in this article.

[0129] Figure 11 The operation of a single magnetic component embodiment of the particle concentration and separation apparatus as described herein is depicted.

[0130] Figure 12 The operation of an angled inlet channel implementation scheme for a particle concentration and separation apparatus as described herein is depicted.

[0131] Figure 13 The process of flow fractionation of particulate samples according to the operation scheme of the particulate concentration and separation apparatus described herein is described.

[0132] Figure 14 The inlet channel magnetic field-assisted sample concentration of an embodiment of the particle concentration and separation apparatus described herein is depicted.

[0133] Figure 15 The inlet channel magnetic field-assisted sample concentration of an embodiment of the particle concentration and separation apparatus described herein is depicted.

[0134] Figure 16 The invention describes a meniscus-assisted concentration of particulate samples according to an operational implementation scheme of the particle concentration and separation apparatus described herein.

[0135] Figure 17 These are micrographs of blood cells separated from a blood sample using an embodiment of the particle concentration and separation apparatus as described herein.

[0136] Figure 18 This is a perspective view of an embodiment of the particle concentration and separation apparatus as described herein.

[0137] Figure 19 is a view of an exemplary flow chamber cartridge showing an imaging surface on a longitudinal side and an illumination surface on a second longitudinal side.

[0138] Figure 20 is a detailed view of an exemplary flow chamber cartridge showing the top and bottom sides of a planar substrate.

[0139] Figure 21 shows an exemplary configuration of the collection hole.

[0140] Figure 22 shows an example of the system receiving block and the orientation of the exemplary flow chamber relative to multiple magnetic components.

[0141] Figure 23 An example of an optical system oriented with an exemplary receiving block is shown.

[0142] Figure 24 Images illustrating exemplary particle separation and outlet channel pressure stability are shown.

[0143] Figure 25 shows examples of collected cell viability and live cell yield.

[0144] Figure 26 shows examples of live cell purity and yield for various cell types collected.

[0145] Figure 27 Microscopic images of unclassified (A, C) and classified (B, D) cell nuclei derived from lung (A, B) and brain (C, D) tissues are shown.

[0146] Figure 28 Flow cytometry analysis of unclassified (A) and classified (B) cell nuclei from Jurkat cells is shown.

[0147] Figure 29 The levels of nucleus-specific targets (SCARNA5) and contaminating cytoplasmic targets (SNHG6) in the isolated samples were shown compared to those in the input cell nuclei isolated by the Levicell system.

[0148] Figure 30 The effect of WGA on bulk cell nucleus separation is shown.

[0149] Figure 31 Images of the LeviCell flow chamber during cell nucleus separation are shown, with and without 3-micron streptavidin-coated polystyrene microbeads.

[0150] Figure 32Images of the top and bottom gradations of the cell counting system collected from LeviCell are shown.

[0151] Figure 33 Spectral data of prestained cell nuclei with and without microbeads are shown.

[0152] Figure 34 The image shows a Seurat rearrangement diagram of a sequenced cell nuclear sample. Detailed Implementation

[0153] I. Definition / Naming

[0154] The following definitions are provided to aid in understanding the invention. Unless otherwise defined, all technical terms, symbols, and other scientific or engineering terms used herein should be understood in the manner commonly understood by those skilled in the art. In some instances, for clarity and / or ease of reference, terms with their commonly understood meanings are defined herein, and these definitions should not be considered materially different from their commonly understood meanings, but rather as supplementary to such commonly understood meanings. Unless otherwise expressly stated, where the definitions herein are inconsistent with those commonly understood in the art, both the definitions provided herein and those commonly understood in the art should be considered within the scope of the invention as alternative embodiments.

[0155] Unless otherwise stated, the open-ended terms used in this article, such as “containing” and its grammatical variations, “incluing” and its grammatical variations, refer to “comprising”.

[0156] Some implementations described herein consider numerical ranges. When a numerical range is provided, it includes the range endpoints unless otherwise specified. Unless otherwise indicated, a numerical range includes all values ​​and subranges therein, as if explicitly stated.

[0157] As used in this article, the article “one” refers to one or more unless otherwise explicitly stated.

[0158] Some values ​​in this document are modified by the term "about". In some instances, the term "about" associated with a reference value may include a range of values ​​plus or minus 10% of that value. For example, the quantity "about 10" may include quantities from 9 to 11. In other embodiments, the term "about" associated with a reference value may include a range of values ​​plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. When a series of values ​​begins with the term "about", the term is intended to modify each value included in the series.

[0159] As used herein, the term “asymmetry” in relation to a magnetic field refers to the magnetic field in the relevant fluid channel region being asymmetrical about one or more planes passing through the center of the fluid channel, and, according to a preferred embodiment, asymmetrical about the horizontal plane.

[0160] As used herein, the term “capillary” or “capillary tube” refers to a tube having a channel as defined below.

[0161] As used herein, the terms “channel,” “flow channel,” “fluid channel,” and “fluidic channel” are used interchangeably and refer to a path in a fluid device in which fluid can flow. The maximum height dimension of the path included in a channel is approximately 100 mm, approximately 50 mm, approximately 30 mm, approximately 25 mm, approximately 20 mm, approximately 15 mm, approximately 10 mm, approximately 5 mm, approximately 5 mm, approximately 3 mm, approximately 2 mm, approximately 1 mm, or approximately 0.5 mm. The dimensions of a channel between magnets are approximately 30 mm × 0.5 mm, approximately 25 mm × 1 mm, approximately 20 mm × 2 mm, approximately 15 mm × 3 mm, approximately 10 mm × 5 mm, approximately 5 mm × 3 mm, approximately 3 mm × 2 mm, approximately 2 mm × 1 mm, or approximately 1 mm × 0.5 mm. For example, the dimensions of a channel between magnets are approximately 2 mm × 1 mm. The internal height of a channel may be non-uniform across its cross-section, and geometrically, the cross-section may be of any shape, including circular, square, elliptical, rectangular, or hexagonal. The term "channel" includes, but is not limited to, microchannels and nanochannels, and for any channel mentioned herein, such channels may include microchannels or nanochannels.

[0162] The term “concentration” as used herein refers to the amount of the first component contained within the second component, and may be based on the number of particles per unit volume, the molar amount per unit volume, the weight per unit volume, or the volume of the first component in the combined components per unit volume.

[0163] As used herein, the terms “fluidically coupled” or “fluidic communication” refer to the ability of fluid to flow between two components that are so coupled or connected.

[0164] As used herein, the terms “isolate” and its grammatical variations, or “separate” and its grammatical variations, or “segregate” and its grammatical variations are used interchangeably, and when referring to a component, they mean the separation of such a component from other components, including increasing the concentration of the component in solution, or separating a component from other components in solution, or a combination of both, i.e., increasing the concentration of the component in solution while separating such a component from other components in solution. A particle in solution is considered “isolated” if it is separated from other particles in solution and / or located within a defined portion of the solution. A particle or component is also considered “isolated” if, after treatment of the solution, the concentration of the particles or component in the solution increases by a ratio of at least about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, or 2:1. If, after treating such a solution, the ratio of the concentration of the target particle to the concentration of other particles increases, or if the ratio of the concentration of the target particle to the concentration of other particles increases by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or if the concentration of other components decreases to less than about 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%, then such target particles in a solution containing such other particles are considered “separated”.

[0165] As used herein, the term "fluid" refers to a system, apparatus, or element used for handling, processing, spraying, and / or analyzing fluid samples, including at least one "channel" as defined above. The term "fluid" includes, but is not limited to, microfluidics and nanofluidics.

[0166] As used herein, the term “fluid function” means any operation, function or process performed or expressed on or expressed in a fluid or sample in a fluid system, including but not limited to filtration, pumping, fluid flow regulation, and control of fluid flow.

[0167] As used herein, the term "particle" refers to any substance, including but not limited to atoms, chemical elements, molecules, compounds, biomolecules, cells, necrotic cells, apoptotic cells, cancer cells, circulating cancer or tumor cells, cell nuclei, blood, plasma, proteins, lipids, body fluids, nucleic acids, nucleotides, amino acids, peptides, antibodies, antigens, carbohydrates, microorganisms, bacteria, viruses, fungi, sperm, gametes, eggs, embryos, or any physical substance whose maximum size in any orientation is less than about 3 mm, 2 mm, 1 mm, 0.5 mm, 0.25 mm, 100 micrometers, 75 micrometers, 50 micrometers, 40 micrometers, 30 micrometers, 20 micrometers, 10 micrometers, 5 micrometers, 2 micrometers, 1 micrometer, or 0.1 micrometers. The maximum size of a particle in any orientation can be from about 0.001 micrometers to about 3 mm, from about 0.1 micrometers to about 2 mm, from about 0.5 micrometers to about 1.5 mm, from about 10 micrometers to about 1 mm, or from about 20 micrometers to about 100 micrometers.

[0168] As used herein, the term "port" refers to a structure used to provide fluid communication between two elements using, for example, a fluid channel. The terms "inlet port," "inlet opening," "input opening," or "input channel" are used interchangeably, and they refer to an opening for injecting sample fluid into the apparatus described herein.

[0169] As used herein, the term "concentrate" and its grammatical variations refer to the process of removing water, aqueous or non-aqueous media, or other substances to achieve a higher bulk density or a purer purity of a substance in a medium. This substance is a type of particle or mixture of particles as described herein. Typically, concentration as described herein involves promoting the sedimentation of particles or a mixture of particles in a medium, thereby carrying the particles or mixture of particles to a specific area. Alternatively, concentration may include separating a specific type of particle from the particle mixture and collecting the specific type of particle in a collection channel, typically with a predetermined volume of liquid medium. Concentration does not require rotating or turning a large quantity of sample to concentrate the particles. Concentration performed by the present invention allows for the separation of particles without significant particle damage, dissolution, or shearing. Additionally, under certain operating conditions, the present invention provides for the separation of flocculation or crystallization within a sample during operation and the separation of flocculated or crystalline particles in the sample.

[0170] When the methods and steps described herein indicate that certain events occur in a specific order, those skilled in the art will recognize that the order of certain steps can be modified, and such modifications are consistent with variations of the invention. Furthermore, certain steps can be performed simultaneously in parallel processes where possible, or they can be performed sequentially.

[0171] II. Magnetic Field

[0172] This disclosure provides a method and apparatus for concentration using a magnetic field within a processing channel or inlet channel. The interaction between the magnetic field and the paramagnetic properties of particles in the sample fluid can provide repulsive or attractive forces to the particles, thereby facilitating separation or concentration.

[0173] According to the embodiments, the magnet is a permanent magnet or an electromagnet. According to the embodiments, the maximum energy product of the magnet ranges from about 1 mega-gauss ostensibly to about 1000 mega-gauss ostensibly, and more preferably from about 10 mega-gauss ostensibly to about 100 mega-gauss ostensibly. According to the embodiments, the surface field strength of the magnet ranges from about 0.1 Tesla to about 100 Tesla, and more preferably from about 1 Tesla to about 10 Tesla. According to the embodiments, the remanence of the magnet ranges from about 0.5 Tesla to about 5 Tesla, and more preferably from about 1 Tesla to about 3 Tesla.

[0174] According to a preferred embodiment, the magnet is made of a material including alloys of neodymium with iron and boron, neodymium, alloys of aluminum with nickel, alloys of neodymium with iron, alloys of aluminum and cobalt with iron, samarium-cobalt, alloys of other rare earth elements with iron, alloys of rare earth elements with nickel, ferrite, or combinations thereof. According to an embodiment including multiple magnets, the magnets may be made of the same material or different materials.

[0175] According to one embodiment, an asymmetric magnetic field is achieved by using a stronger magnetic material on one side of the fluid channel and a weaker magnetic material on the opposite side. According to a preferred embodiment, an asymmetric magnetic field is achieved by using a magnetic material on one side of the fluid channel and a substantially similar magnetic material on the opposite side. According to such embodiments, the upper and lower magnets can have substantially the same size. According to such embodiments, the upper magnet may comprise neodymium, the lower magnet may comprise samarium-cobalt, and the two magnets are substantially the same size. Alternatively, the upper magnet may comprise samarium-cobalt, the lower magnet may comprise neodymium, and the two magnets are substantially the same size.

[0176] According to the embodiments, alternative magnet configurations can be used. A device according to the invention may include a plurality of upper magnets and a plurality of lower magnets positioned around a fluid channel. The upper magnets may include a front upper magnet, a central upper magnet, and a rear upper magnet. The lower magnets may include a front lower magnet, a central lower magnet, and a rear lower magnet.

[0177] According to another magnet configuration, the device may include a front upper magnet, a rear upper magnet, a front lower magnet, and a rear lower magnet, wherein the magnets are positioned around the fluid channel. The front upper magnet and the rear lower magnet are positioned in a magnetic repulsion direction. Exemplary NdFeB magnetic assembly dimensions include approximately 50 × 15 × 2 mm for the bottom magnetic assembly (magnetized via a 15 mm axis) and 50 × 5 × 2 mm for the top magnetic assembly (magnetized via a 5 mm axis). Other magnet assembly embodiments include 60 × 15 × 2 mm, 60 × 5 × 2 mm, 75 × 20 × 3 mm, and 25 × 15 × 2 mm. Figure 6 An embodiment with rectangular magnets substantially aligned along the X-axis at the bottom of processing channel 104 is shown, illustrating the magnetic field lines and magnetic force within the processing channel. Another preferred embodiment of the magnet assembly includes an upper magnet and a lower magnet with dimensions of approximately 75 × 20 × 3.2 mm, and the spacing between the upper and lower magnets is approximately 2.5 mm, approximately 3.0 mm, approximately 3.5 mm, approximately 2.9 mm, approximately 3.0 mm, approximately 3.1 mm, approximately 3.2 mm, approximately 3.3 mm, or approximately 2.72 mm, approximately 2.88 mm, approximately 2.98 mm, approximately 3.18 mm, approximately 3.20 mm, or approximately 3.37 mm.

[0178] In a preferred embodiment, the device has an upper magnet and a lower magnet, wherein the flower magnet extends into the inlet channel. The bottom magnet dimensions can be from about 50 mm to about 100 mm × about 10 mm to about 30 mm × about 2 mm to about 4 mm. Preferred embodiments include about 75 mm, about 80 mm, about 85 mm, about 90 mm, about 93 mm, or about 95 mm × about 15 mm, about 18 mm, about 20 mm, about 23 mm, and about 25 mm × about 2 mm, about 2.3 mm, about 2.5 mm, about 2.7 mm, about 3 mm, about 3.18 mm, and about 3.5 mm. The magnet spacing between the upper and lower magnets is preferably 2 to 4.3 mm, about 2.5 mm, about 4.0 mm, about 3.5 mm, about 2.9 mm, about 3.0 mm, about 3.1 mm, about 3.2 mm, about 3.3 mm, or about 2.72 mm, about 2.88 mm, about 2.98 mm, about 3.18 mm, about 3.20 mm, about 3.37 mm, about 3.5 mm, about 3.7 mm, or about 4 mm.

[0179] Figure 7 The AD shows an embodiment of a fluid concentration device in which parallel magnet assemblies are combined at the top and bottom and are substantially aligned along the X-axis of the processing channel.

[0180] III. Paramagnetic media

[0181] Samples processed by magnetically enhanced concentration according to the present invention typically contain added paramagnetic or diamagnetic components. According to the method of the present invention, a substance containing target particles is mixed with a paramagnetic medium to produce a processing solution. The paramagnetic medium comprises a paramagnetic material and a solvent. According to a preferred embodiment, the paramagnetic medium is biocompatible, i.e., capable of mixing with living cells without affecting cell viability or cell behavior, such as affecting gene expression. The paramagnetic material may be selected from gadolinium, titanium, vanadium, dysprosium, chromium, manganese, iron, nickel, gallium, including their ions and combinations thereof. According to an embodiment, the paramagnetic material is selected from titanium(III) ions, gadolinium(III) ions, vanadium(I) ions, nickel(II) ions, chromium(III) ions, vanadium(III) ions, dysprosium(III) ions, cobalt(II) ions, and gallium(III) ions. According to a preferred embodiment, the paramagnetic material comprises a chelating compound. According to a preferred embodiment, the paramagnetic material comprises a gadolinium chelate, a dysprosium chelate, or a manganese chelate. According to the embodiments, the paramagnetic medium comprises a paramagnetic material, a salt, and other additives that maintain cell integrity. In embodiments of the present invention, the paramagnetic material may be [Aliq]2[MnCl4], [Aliq]3[GdCl6], [Aliq]3[HoCl6], [Aliq]3[HoBr6], [BMIM]3[HoCl6], [BMIM][FeCl4], [BMIM]2[MnCl4], [BMIM]3[DyCl6], [BDMIM]3[DyCl6], [AlaCl][FeCl4], [AlaCl]2[MnCl4], [AlaCl]3[GdCl6], [AlaCl]3[HoCl6], [AlaCl]3[DyCl6], [GlyC2][FeCl4], as described in U.S. Patent Application Serial No. 14 / 407,736, which is incorporated herein by reference.

[0182] According to the embodiments, the paramagnetic material can be present in the paramagnetic medium at concentrations of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 150 mM, 200 mM, 250 mM, 300 mM, 500 mM, or 1 M. According to the embodiments, the paramagnetic material can be present in the paramagnetic medium at concentrations of about 10 mM to about 50 mM, about 25 mM to about 75 mM, about 50 mM to about 100 mM, about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 500 mM, or about 500 mM.

[0183] According to an embodiment, the paramagnetic material comprises gadolinium and is present in the paramagnetic medium at a concentration of at least about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. According to an embodiment, the paramagnetic material comprises gadolinium and is present in the paramagnetic medium at a concentration of about 10 mM to about 50 mM, about 25 mM to about 75 mM, or about 50 mM to about 100 mM.

[0184] IV. Equipment Configuration

[0185] See Figures 1 to 5Figures 7-10 illustrate various embodiments of the particle concentration apparatus of the present invention for particle concentration and separation, wherein the inlet channel, processing channel, and outlet channel portions of the apparatus comprise interconnected separate individual components. The processing channel is preferably an elongated fluid channel having a sufficient length along the x-axis to allow sufficient time for processing the fluid containing the target particles, said processing time being based on the residence time required for particle concentration within a layer of flow within the processing channel (where "layer" in this sense indicates a small range of location along the y-axis) and based on the expected throughput from the system. In some embodiments, the processing channel is a fluid channel with a height of approximately 200 micrometers to approximately 30 mm, approximately 200 micrometers to approximately 20 mm, approximately 200 micrometers to approximately 15 mm, approximately 200 micrometers to approximately 10 mm, approximately 200 micrometers to approximately 5 mm, approximately 200 micrometers to approximately 2 mm, approximately 200 micrometers to approximately 1 mm, approximately 0.5 mm to approximately 1 mm, approximately 0.5 mm to approximately 2 mm, approximately 0.5 mm to approximately 3 mm, approximately 1 mm to approximately 2 mm, approximately 1 mm to approximately 3 mm, or approximately 1.5 mm to approximately 2 mm. According to the embodiments, the length of the processing channel is approximately 20 mm to approximately 200 mm, approximately 20 mm to approximately 150 mm, approximately 20 mm to approximately 100 mm, approximately 20 mm to approximately 50 mm, approximately 40 mm to approximately 100 mm, approximately 40 mm to approximately 90 mm, or approximately 40 mm to approximately 80 mm. For some embodiments, the channel depth (in the Z direction) is approximately 100 micrometers to approximately 5 mm, approximately 500 micrometers to approximately 3 mm, approximately 1 mm to approximately 2.5 mm, or approximately 1.5 to approximately 2 mm. Channel length embodiments can be 40, 45, 50, 60, or 70 mm long. Exemplary processing channel dimensions include a height of 1 mm and 1.9 mm (Y-axis, vertical direction in the image) × a depth of 0.8, 1.0, 1.5, or 2.0 mm (Z-axis), and a length (X-axis) of 40-70 mm. A preferred embodiment of the processing channel has exemplary processing channel dimensions comprising approximately 2 mm height (Y-axis, vertical direction in the image) × approximately 2.0 mm depth (Z-axis) and a length (X-axis) of approximately 50-70 mm. Further preferred embodiments of the processing channel have lengths of approximately 55 mm, approximately 56 mm, approximately 57 mm, approximately 58 mm, approximately 59 mm, approximately 60 mm, approximately 61 mm, approximately 62 mm, approximately 63 mm, approximately 64 mm, approximately 65 mm, or approximately 66 mm.

[0186] The processing channel can have any cross-sectional geometry and may include square, rectangular, circular, or elliptical cross-sectional geometries. The geometric features of the processing channel described herein also apply to the inlet, outlet, and any other fluid channels configured with reference to the components described above.

[0187] The cross-sectional area of ​​the inlet channel (when the channel is circular, it will be πr) 2 The diameter (where r is the radius of the channel's inner diameter) is substantially smaller than the cross-sectional area of ​​the processing channel. In this context, "diameter" is used to describe the characteristic cross-sectional size, and the channel's cross-section may not be circular. In various embodiments, the cross-sectional area of ​​the inlet channel is at least 100 times, 80 times, 50 times, 40 times, 20 times, 10 times, 8 times, 6 times, 4 times, or 2 times smaller than the cross-sectional area of ​​the processing channel. In some embodiments, the cross-sectional area of ​​the inlet channel is at least 10 times smaller than the cross-sectional area of ​​the processing channel. In some embodiments, the cross-sectional area of ​​the inlet channel is at least 5 times smaller than the cross-sectional area of ​​the processing channel. In some embodiments, the cross-sectional area of ​​the inlet channel is at most 0.2 mm. 2 At most 0.8 mm 2 At most 3.1 mm 2 At most 7.1 mm 2 At most 12.6 mm 2 At most 19.6 mm 2 At most 28.3mm 2 At most 38.5 mm 2 At most 50.3 mm 2 At most 78.5 mm 2 At most 176.7 mm 2 Or at most 314.2 mm 2 The exit channel typically has similar dimensional characteristics to the inlet channel, but may have varying cross-sectional areas as described below.

[0188] Embodiments of the particle concentration apparatus include a tapered inlet portion of the processing channel (103). The particle separation apparatus of the present invention also includes a tapered inlet port to reduce turbulence caused by eddies, and thus reduce shear forces associated with the connection of fluid channels with different cross-sectional areas. These eddies can reduce the efficiency or rate of sample processing because they can create locations where cells or other particles become trapped in the circulation path and do not flow through the apparatus. Eddies can also exert shear stress on particles such as cells. The cone angle can be between about 10° and about 70°, preferably about 20° to about 60°, about 30° to about 45°, or in some embodiments, about 30°.

[0189] The outlet portion of the device may include a diverter that helps to split a portion of the sample flow in the processing channel into discrete flows for separation or further processing. The diverter is preferably positioned within the processing channel, but near the tail end of the processing channel, such that any particle separation achieved by passing the fluid through the processing channel is maintained as the fluid exits the device. The diverter may include one or more horizontal baffles extending from the outlet channel into the tail end of the processing channel. In embodiments where the magnets are substantially aligned along the X-axis and located on opposite sides of the processing channel, the diverter may extend into the processing channel with a length of 0.5–3.5 x, 1–3 x, 1.5–2.5 x, 1–2 x, or 2 x the distance between the magnets. For single-magnet embodiments of the particle concentration device, the diverter may extend into the processing channel with an extension thickness of 1–5 x, 1.5–5 x, 1.5–4 x, 2–4 x, 3–4 x, or 4 x the thickness of the individual magnet assembly in the Z-axis direction. For embodiments of magnet assemblies not aligned along the X-axis of the processing channel, the diverter may extend between 5-40%, 5-30%, 5-25%, 10-30%, 10-20%, or 10-15% of the processing channel length, preferably greater than 5%, and preferably less than 35%. The diverter tapers to point towards the end within the processing channel. The cone angle may be about 5° to about 45°, preferably about 10° to about 30°, about 15° to about 25°, or in some embodiments, about 20°. The diverter may be used to horizontally divide the processing channel. Furthermore, the diverter may include one or more vertical baffles, thereby forming a horizontal and vertical grid of outflow fluid outlet openings that are fluidly connected, leading to multiple outlet channels. In this embodiment, multiple outlet channels near the end of the processing channel lead to multiple collection chambers, such as collection tubes or Eppendorf tubes, via outlet ports. The diverter defines multiple outlet channels. According to an embodiment, the particle concentration apparatus of the present invention includes a diverter defining 2, 3, 4, 5, 6, 7, 8, 9, or 10 outlet channels. According to an embodiment, the particle concentration apparatus of the present invention includes a diverter defining at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 outlet channels. According to an embodiment, the particle concentration apparatus of the present invention includes a diverter defining 2 to 4, 5 to 7, or 8 to 10 outlet channels. The diverter described herein and the various outlet channels resulting therefrom can be incorporated into the processing channels described above in the component configuration of the present invention.

[0190] Multiple outlet channels extend from the processing channel to corresponding outlet ports. These outlet channels may include fluid flow regulators, such as pumps or output valves, that control the amount of flow from the processing channel through the corresponding outlet channels to the corresponding outlet ports. Distributing the sample solution into each effluent fraction can be achieved by increasing or decreasing the fluid flow rate to each outlet, thereby adjusting the distribution ratio. According to embodiments, this ratio can be adjusted by up to 50%. For example, if the distributor contains two channels with equal cross-sections, the geometric distribution ratio is 1:1. The distribution ratio can be adjusted, for example, to about 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, by using a larger (or smaller) pumping rate than the other fraction to introduce a larger (or smaller) amount of fluid into one fraction. In a preferred embodiment, this geometric distribution ratio ranges from about 2:1 to about 1:2. In other preferred embodiments, this geometric distribution ratio ranges from about 10:1 to about 1:10. The velocity distribution ratio between the upper outlet channel and the lower channel can be approximately 1:10, 1:9.5, 1:9, 1:8.5, 1:8, 1:7.5, 1:7, 1:6.5, 1:6, 1:5.5, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5:5, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1.

[0191] Figure 1 A shows an apparatus having a single magnet assembly (101) substantially aligned along the top X-axis of a processing channel (104), an inlet channel (102) and an inlet connection region 103, multiple outlet channels (106) and a flow diverter section (105). Figure 1 B shows a similar configuration, where 101 is aligned along the bottom of the processing channel. Figure 1 C shows Figure 1 The configuration in A, wherein an additional magnet component or portion (107) extends beyond the processing channel and enters the inlet portion. Figure 1Figure D illustrates this type of configuration, in which the magnet assembly is substantially aligned along the X-axis at the bottom of the processing channel and extends into the inlet portion. According to embodiments, one or both of the upper and lower magnets are movably mounted within the system to allow controlled adjustment of the magnets' vertical position relative to the fluid channel, thereby modulating the magnetic field strength within the channel. The use of the inlet channel geometry and the inlet channel magnetic field is employed in some embodiments of the invention to modulate particle concentration within the processing channel. By accumulating particles at or near the inlet of the processing channel, some embodiments are able to create a portion of the flow stream that varies in terms of relative particle enrichment. For example, particle accumulation at or near the inlet of the processing channel can result in an instantaneous removal of the amount of particles from the flow stream within the processing channel. By releasing or overcoming particle-binding forces, deposition forces, magnetic forces, or combinations thereof, the particle concentration within the flow stream in the processing channel subsequently increases. Over time, this can lead to changes in the particle enrichment level within the particle-rich layer of the flow stream in the processing channel. In one aspect, this selective process is useful in flow stream components of particular interest to users of the apparatus and methods of the present invention, who can identify and utilize particularly enhanced (enriched) or particularly removed flow stream components.

[0192] Figure 2 AD illustrates a single-magnet assembly configuration, further comprising an inlet channel portion (203) having a portion substantially linearly aligned with the processing channel and a portion nonlinearly aligned with the processing channel, the two portions being joined at a portion at an angle θ. The nonlinear inlet portion may contain one or more cross channels (201), and the linear inlet portion may contain one or more cross channels (202). The cross channels of the inlet channel provide for the introduction or removal of liquids, suspensions, or gases. The cross channels can be used to introduce reagents into the sample flow, such as paramagnetic media, buffers, flocculants, sample pretreatment reagents, or reactants. The cross channels can also be used to introduce reaction reagents into the sample medium for reaction with particles or other components of the sample medium within the inlet channel or the processing channel. For example, cell staining reactions or ligand binding reactions may be performed in the inlet channel prior to separation. Additionally, in some embodiments, reactions may be performed or continued in the processing channel, such as in the case of precipitation, flocculation, or crystallization reactions, if necessary or desired. One or more cross channels may also be used as outputs to collect particles that are repelled or otherwise prevented from entering the separation channel. Figure 2 A illustrates a fluid concentrator having a magnet assembly substantially aligned along the X-axis at the top of the processing channel. The nonlinear portion of the inlet channel is at approximately 90° relative to the substantially linear portion of the inlet channel. Figure 2B illustrates a similar implementation in which the magnet assembly is substantially linearly aligned at the bottom of the processing channel, and the non-linearly aligned portion of the inlet channel is at approximately 270° relative to the substantially linear portion of the inlet channel. Figure 2 C shows, as Figure 2 In the implementation scheme of A, the magnet assembly is aligned along the bottom of the processing channel. Figure 2 D shows a similar Figure 2 In implementation B, the magnet assembly is aligned along the bottom of the processing channel. Figure 2 In the AD implementation scheme, the non-linearly aligned inlet channel portion is approximately 90° relative to the fundamentally linear portion of the inlet channel. Figure 2 B, 2D) or approximately 270° ( Figure 2 (A, 2C) The angle θ can be determined according to the device requirements, where θ ≠ 0°, θ ≠ 180°, and any angle θ relative to the X-axis and / or independently relative to the Y-axis and Z-axis, θ ≥ 30°, ≥ 45°, ≥ 70°, ≥ 90°, ≥ 100°, ≥ 135°, ≥ 140°, ≥ 165°, 180°, ≥205°, ≥225°, ≥250°, ≥280°, ≥300°, or ≤330°. For example, Figure 2 E shows a planar graph where θ(x) = 90°, θ(y) = 180°, and θ(z) = 0°. Figure 2 F shows a planar graph where θ(x) = 90°, θ(y) = 225°, and θ(z) = 0°.

[0193] Figure 3 AD shows the comparison with Figure 2 The AD implementation is similar to the implementation in which the magnet assembly 107 extends into the inlet region.

[0194] Figure 4 AD illustrates an embodiment of a fluid concentration apparatus with another component (401), which is an annular or ring-shaped magnet assembly (ring magnet) surrounding an inlet channel. The ring magnet may include one or more magnets positioned in a repulsive direction relative to their magnetic poles, thereby generating a symmetrical magnetic force along the X-axis of the inlet channel. In one embodiment, the ring magnet includes a plurality of rectangular magnets configured around the inlet such that the respective repulsive fields are aligned within the inlet channel. The force applied from the ring magnet slows the particle flow rate by providing a repulsive force against the paramagnetic properties of the particles. The ring magnet may be configured in a substantially linear portion of the inlet channel, such as... Figure 4 As shown in Figure A, the magnet assembly is substantially aligned along the X-axis at the top of the processing channel, and as... Figure 4As shown in B, the magnet assembly is essentially aligned along the X-axis at the bottom of the processing channel. Figure 4 C shows Figure 4 The configuration in A, wherein an additional magnet component or portion (107) extends beyond the processing channel and enters the inlet portion. Figure 4 D shows an annular magnet arranged around the inlet portion of a fluid concentrator, wherein the magnet assembly is not aligned along the axis of the processing channel. Although as... Figure 4 The 401 shown in AD is a linear portion surrounding the inlet channel, but these embodiments are not limiting, and the toroidal magnet can be placed anywhere along the inlet channel in other embodiments, including non-linearly aligned portions of the inlet channel.

[0195] Figure 5 AD illustrates an embodiment having a single magnet assembly substantially aligned at the top or bottom along the X-axis of the processing channel, and also includes a toroidal magnet 401 and a non-linearly aligned inlet channel portion as well as cross channels. One or more cross channels may also be used as an output to collect particles that are repelled or otherwise prevented from entering the separation channel.

[0196] Many implementations of particle concentration devices include a single magnet assembly aligned substantially at the top or bottom of the X-axis of the processing channel. Other implementations described herein do not have a magnet assembly aligned along most of the processing channel. In other implementations, the device may include multiple magnet assemblies aligned substantially at the top and bottom of the X-axis of the processing channel. These implementations achieve magnetic levitation of paramagnetic particles within the channel, thereby separating heterogeneous particles within the processing channel. According to the implementation, the levitation device includes an upper or lower magnet comprising multiple movable magnets, thereby controlling the number of magnets involved in the operation (i.e., actively generating a magnetic field within the processing section of the fluid channel), and thus controlling the strength and gradient distribution of the magnetic field. Control of the magnetic field over time enables more complex experimental schemes that can be modified at any time during the experiment or analysis. This scheme offers several advantages over static systems, such as: more flexible sample separation; higher particle separation resolution; more flexible fluid path cleaning, pretreatment, and processing methods; and providing feedback during the experiment or analysis to optimize or modify separation parameters.

[0197] According to one embodiment, the upper and lower magnets comprise elongated rectangular magnets (preferably bar magnets) with dimensions ranging from approximately 2 mm to approximately 25 mm in height (y-axis direction (vertical axis)), approximately 30 mm to approximately 80 mm or approximately 95 mm in width (x-axis direction), and approximately 0.5 mm to approximately 7 mm in depth (z-axis direction). Preferably, the dimensions of the upper and lower magnets range from approximately 4 mm to approximately 20 mm in height (y-axis direction), approximately 40 mm to approximately 60 mm in width (x-axis direction), and approximately 1 mm to approximately 3 mm in depth (z-axis direction). The preferred magnet dimensions described herein can be achieved by a single magnet or a combination of multiple magnets. According to the embodiment, the depth and width of the upper and lower magnets are substantially the same. According to one embodiment, the height of the upper magnet is at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% greater than the height of the lower magnet. According to another embodiment, the height of the upper magnet is about 25% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, or about 500% to about 600% greater than the height of the lower magnet. According to one embodiment, the height of the lower magnet is at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% greater than the height of the upper magnet. According to another embodiment, the height of the lower magnet is about 25% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, or about 500% to about 600% greater than the height of the upper magnet.

[0198] According to the embodiment, the distance along the vertical axis between the upper and lower magnets and the fluid channel, capillary, or central processing section is at least about 1 micrometer, 10 micrometers, 50 micrometers, or 100 micrometers and / or no more than about 500 micrometers, 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm. According to the embodiment, the distance along the vertical axis between any one of the magnets and the fluid processing channel is about 1 micrometer to about 5 mm, and preferably about 10 micrometers to about 2 mm.

[0199] According to the implementation scheme, the vertical distance between the upper magnet and the fluid processing channel is at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% greater than the vertical distance between the lower magnet and the fluid processing channel. According to the implementation scheme, the vertical distance between the upper magnet and the fluid processing channel is at least about 25% greater than the vertical distance between the lower magnet and the fluid processing channel to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, or about 500% to about 600%.

[0200] According to the implementation scheme, the vertical distance between the lower magnet and the fluid processing channel is at least about 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% greater than the vertical distance between the upper magnet and the fluid processing channel. According to the implementation scheme, the vertical distance between the lower magnet and the fluid processing channel is at least about 25% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, or about 500% to about 600% greater than the vertical distance between the upper magnet and the fluid processing channel.

[0201] According to the embodiment, the upper and lower magnets are permanent magnets or electromagnets. According to the embodiment, the maximum energy product of the upper and lower magnets ranges from about 1 mega-gauss ostensons to about 1000 mega-gauss ostensons, and more preferably from about 10 mega-gauss ostensons to about 100 mega-gauss ostensons. According to the embodiment, the surface field strength of the upper and lower magnets ranges from about 0.1 Tesla to about 100 Tesla, and more preferably from about 1 Tesla to about 10 Tesla. According to the embodiment, the remanence of the upper and lower magnets ranges from about 0.5 Tesla to about 5 Tesla, and more preferably from about 1 Tesla to about 3 Tesla.

[0202] According to a preferred embodiment, an asymmetric magnetic field is achieved by using a stronger magnetic material on one side of the processing channel and a weaker magnetic material on the opposite side of the processing channel. According to a preferred embodiment, an asymmetric magnetic field is achieved by using a magnetic material on one side of the fluid channel and a substantially similar magnetic material on the opposite side of the fluid channel. According to such embodiments, the upper and lower magnets can have substantially the same size. According to such embodiments, the upper magnet may comprise an alloy of neodymium and iron, and the lower magnet may comprise samarium-cobalt, wherein the two magnets are substantially the same size. Alternatively, the upper magnet may comprise samarium-cobalt, and the lower magnet may comprise neodymium, wherein the two magnets are substantially the same size.

[0203] According to the embodiments, alternative magnet configurations can be used. A device according to the invention may include a plurality of upper magnets and a plurality of lower magnets positioned around a fluid channel. The upper magnets may include a front upper magnet, a central upper magnet, and a rear upper magnet. The lower magnets may include a front lower magnet, a central lower magnet, and a rear lower magnet.

[0204] According to another magnet configuration, the device may include a front upper magnet, a rear upper magnet, a front lower magnet, and a rear lower magnet, wherein the magnets are positioned around the fluid channel. The front upper magnet and the rear lower magnet are positioned in a direction of magnetic repulsion. An asymmetric magnetic levitation device is further described in international patent application PCT / US19 / 24138, which is incorporated herein by reference.

[0205] Figure 7 AD illustrates an embodiment of a dual-magnet assembly particle concentration apparatus having an optional annular magnet 401 with an exemplary orientation and optional inlet channel portions 201, 202, and 203.

[0206] Some embodiments of the particle concentration apparatus described herein do not have a magnet assembly aligned along most of the processing channel. Particle concentration is achieved by sedimentation and optionally by a combination of pre-concentration in the inlet channel by overcoming magnetic repulsion of the particles and pre-concentration mediated by flow, which may optionally be enhanced by the geometry of the inlet channel. Figure 8 AD illustrates an implementation scheme for the device configuration. Figure 8 EH illustrates incorporating inlet channel valves (801, 802, 803) that control the movement of fluid to optional cross channels. A pump (804) can be associated with any channel, inlet, inlet cross, outlet, deflection, etc. As further described herein, the pump can be configured in various embodiments to provide the required positive or negative pressure to drive fluid and accumulate flow in the corresponding channel when operating among multiple pumps associated with a fluid-communicating channel. Figure 8 H illustrates a particle concentrator with inlet cross channels 805 and 806. (See diagram for reference.) Figure 8As shown in H, the inlet channel and the channel intersection can be located upstream, downstream, or both of the toroidal magnet. In one embodiment, the intersection channel (806) is provided only in the portion of the inlet channel that is substantially in line with the processing channel, and this intersection channel is located upstream of the toroidal magnet.

[0207] Figure 9 illustrates an implementation scheme for a particle concentration device with a single magnet assembly. Figure 9A A particle concentrator apparatus having a pump (804) that drives the flow from the inlet channel to the processing channel is shown. Figure 9B A particle concentration apparatus with multiple pumps (804) is shown, each pump individually controlling the flow rate of the outlet channel. The combined flow rates are additive for the flow in the processing and inlet channels. The flow rates can range from 0.1 μL to 1 mL / min, depending on the sample fluid characteristics, such as viscosity and particle concentration, as well as the size and configuration of the inlet, processing, and outlet channels. In a preferred embodiment, the flow rates in the processing and outlet channels can be from about 0.2 μL / min to about 200 μL / min, or from about 0.5 μL / min to about 40 μL / min. Concentration can also be performed under stopped or intermittent flow operation. A valve (902) can be actuated to redirect the outlet channel to outlet port (903) or (904). In some embodiments, the inlet and outlet channel pumps are configured for positive or negative pressure relative to the processing channel, respectively. Under stopped flow conditions, sample separation is performed, and the sample is moved to the separated outlet channel by restoring flow in the processing channel.

[0208] According to the present invention, the device may include one or more pumps to drive fluid through the device. The term "pump" is used to refer to any device that applies a pressure difference between different locations within the device. The pump may be placed at the inlet side of the system (pushing fluid towards the outlet) or at the outlet (drawing liquid out of the inlet), or a combination of both. The pressure difference may be positive or negative. Pressure differences may be applied concurrently to multiple outlets or inlets, or may be arranged such that each outlet or inlet has a directly applied pressure difference. In some embodiments, the pressure difference may be applied to overcome particle pre-concentration forces, magnetic forces, deposition forces, or combinations of forces in the inlet channel. The pump may be variable to allow control of the applied pressure difference. Types of pumps include, but are not limited to: positive displacement pumps, such as syringe pumps; peristaltic pumps; diaphragm pumps; adjustable hydrostatic sources; gravity-controlled pressure sources, such as increases or decreases in liquid volume; and manual pressure sources, such as plastic or aluminum foil blister packs.

[0209] In some embodiments, a pump may be positioned on an inlet line to generally drive fluid flow through the channel structure; it may also be positioned on certain outlet channels or ports to drive fluid flow through directional outlet lines. For example, a pump may be positioned on one or more outlet lines associated with one or more particle enrichment or particle removal layer heights for one or more target particles. Furthermore, all outlet lines may include variable pumps that can be activated or deactivated based on one or more expected flow heights for one or more target particles. Similarly, an external pump may be controlled to provide a variable pressure differential. Embodiments may also include additional components, including: a container for housing one or more outlet collection tubes; a container for housing one or more inlet tubes; an assembly including a container for housing one or more tubes, the container being temperature-controlled, such as a cooling plate storing one or more outlet tubes at a temperature close to 4°C; or a microplate holder that may include positioning means for connecting an inlet or outlet to a hole in the microplate. In embodiments, the outlet channel or port is fluidly connected to a pipetting robot. The pipetting robot can be integrated with a particle concentrator to selectively dispense aliquots or fractions of concentrated particle groups from the processing channel, and alternatively or additionally, dispense aliquots or fractions of particle-removed effluent from the processing channel. The device can also be integrated with a microprocessor or computer programmed to record, analyze, and / or control fluid and / or particle flow and separation through the device.

[0210] Figure 10 A particle concentration system including an exemplary particle concentration apparatus is shown. The apparatus may also include one or more sensors. For example, a processing channel sensor (1001) and an inlet channel sensor (1002) may be implemented in the system. A system integrator (1003) may include a signal processor to receive signals from sensors (1001) and (1002) and control other components, such as an inlet pump (804) or an outlet channel pump, a magnetic field of a toroidal magnet (201) or other electromagnetic components, valves or other interface devices or components, via pre-programmed or programmable commands. Sensors are selected from optical sensors, capacitive sensors, conductive sensors, thermal sensors, flow rate sensors, ultrasonic sensors, gravity sensors, magnetic field sensors, or combinations thereof. In embodiments, the sensor is a photodetector, a multi-pixel imaging detector, a magnetic field detector, an electrochemical detector, an optical phase detector, a scattering detector, a Hall sensor, a magnetoresistive sensor, a radiation thermal sensor, a surface acoustic wave sensor, a biosensor, or a combination thereof. The sensor may also include one or more sensors located within or near a portion of the processing channel or inlet channel to detect the presence or quantity of particles, or other physical or chemical properties of the particle or sample flow.

[0211] like Figure 18The plan view of the particle concentration apparatus shown may further include a visualization component or imaging sensor, a sensor illuminator, and sensor optics. The visualization component may include any device capable of enabling or enhancing the ability to observe and / or record particles passing through a processing channel in real time, thereby enabling the observation and / or measurement of particle separation, including the degree and / or rate of particle separation. Visualization may also include analyzing the size, shape, or other properties of the particles and / or other components of the sample. According to one embodiment, the material used to surround and thus define the processing channel is transparent or light-transmitting in at least one section of the processing channel to facilitate observation of particles passing through it. The visualization system may employ optics to allow bright-field illumination, dark-field illumination, and / or fluorescence detection of sample components.

[0212] In one embodiment, the device includes two optically transparent or light-transmitting channel segments, each located on opposite sides of a channel. According to this embodiment, a visualization component is positioned on one side and focused through one of the transparent or light-transmitting segments, and an illumination component is positioned on the opposite side and focused through a second transparent or light-transmitting segment. The illumination component is configured to provide sufficient light to facilitate visualization of particles within the processing channel by the visualization component.

[0213] In another embodiment, the device includes a transparent or light-transmitting section on one side of the channel. According to this embodiment, a visualization component is positioned on one side and focused through the transparent or light-transmitting section, and an illumination component is positioned on the same side and focused through the transparent or light-transmitting section. The illumination component is configured to provide sufficient light to facilitate visualization of particles within the processing channel by the visualization component.

[0214] In a preferred embodiment, the illumination system is a visible light source or an ultraviolet light source. The illumination source can be configured to illuminate the sample in an optically transparent flow channel including a processing channel, wherein light is transmitted through the flow channel to an optical sensor opposite the illumination source. In one embodiment, the illumination source is angled adjacent to the optical sensor such that light from the source is reflected from the sample in the flow channel into the optical sensor. In another embodiment, the light source is an ultraviolet light source and is constructed and arranged to illuminate the sample within the flow channel such that visible light from the natural fluorescence of the artificial fluorescence associated with the sample is detected by the optical sensor.

[0215] V. Particle Separation and Concentration Methods

[0216] Organic or inorganic particles can be concentrated using the methods of this invention. Particles can be biological entities such as cells, cell debris, organelles (e.g., cell nuclei), clusters, tissues, tissue components, microorganisms (including bacteria, fungi (yeasts and molds)), viruses, protozoa and algae and their debris, organelles, clusters, and other components. Particles can also be macromolecules, complexes, chelates, conjugates, crystals, amorphous solids, gels, coagulations, etc. DNA, RNA, and proteins can be concentrated using the methods of this invention. Microbeads, shells, nanoparticles, laminates, precipitates, and coprecipitates can also be concentrated. Many applications require particle separation, including applications that require separating similar particles from other particles, identifying particles, and handling or otherwise manipulating particles. These applications include, but are not limited to, separating live and dead cells, separating cell nuclei from live and dead cells and nuclear debris, separating and / or treating circulating tumor cells, emulsion PCR enrichment, generating plasma such as platelet-rich plasma, separating sperm for specific traits such as sex selection, bacterial load testing, antibiotic resistance testing, identification of sepsis or blood contamination, immune cell separation, compound screening, separation of foreign bodies, or separation of extracellular vesicles. The particle separation method of the present invention can be used in any of these applications.

[0217] Particles present in a sample medium are concentrated in a particle concentration apparatus under conditions that substantially enrich the particle concentration and substantially remove the sample medium layer. Sample media with heterogeneous particle clusters can be selectively enriched based on size, density, paramagnetic inhomogeneity and selective orientation of magnetic forces, and the flow rate of the processing channel. Heterogeneous particle clusters may originate from biological samples. In some cases, as illustrative examples, biological samples are bodily fluids, including blood, saliva, urine, sperm, plasma, serum, and feces; swabs, including skin, anal, nasal, and vaginal swabs, or environmental swabs from doorknobs; and proximal fluids, including tears, lavage fluid from the lungs, or interstitial tissue fluid from the breast. In some cases, as illustrative examples, biological samples are live and dead cells, lysed cells, circulating tumor cells, nucleic acids, nucleotides, amino acids, peptides, proteins, antigens, antibodies, or immune cells (e.g., leukocytes, T cells, phagocytes). In some cases, as illustrative examples, biological samples are biomolecules, cells, proteins, lipids, carbohydrates, microorganisms, viral particles, or bacteria.

[0218] The concentration level of the particle enrichment fraction in the sample medium exceeds the particle concentration by at least 30%, preferably 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In one embodiment, the particle removal fraction is substantially free of particles.

[0219] Before being introduced into the processing channel, reagent fluids or gases may be introduced into the flow stream before, simultaneously with, or after the sample medium passes through the location containing the intersecting channels. In one embodiment, paramagnetic media, buffer solutions, flocculants, sample preconditioning reagents, or reactants may be introduced and mixed into the sample medium preparation for concentration, separation, or analysis in the processing channel.

[0220] Particles are introduced into a processing channel at a certain flow rate and undergo sedimentation and / or magnetic repulsion and / or magnetic attraction within the channel to form a particle enrichment and removal layer within the flow stream. In various embodiments, the particle enrichment and removal layer forms within the flow stream under continuous, stopped, or intermittent flow conditions. The particle enrichment and / or removal layers are collected as the layers separate within the flow stream of the processing channel. The flow output, particle enrichment stream, and / or particle removal stream exiting the processing channel can be selectively directed for fractionation or staging. Once the target particles reach their equilibrium (or near-equilibrium) height in the flow layer within the processing channel, they pass through a splitter, distributing the treatment solution into multiple fractions. Because the target particles are geometrically separated in the treatment solution, essentially all target particles are retained in the effluent of some geometric fraction. The geometric effluent fractions containing the target particles are then collected, and if target particles are present in more than one fraction, they can be recombine to separate the target particles. In some embodiments, it may be necessary to separate the cells from the paramagnetic medium. If it is necessary to separate cells from a paramagnetic medium, this can be done by dilution.

[0221] Alternatively, the sample solution can be divided into individual effluent fractions by increasing or decreasing the fluid flow rate to each outlet, thereby adjusting the distribution ratio. According to one embodiment, this ratio can be adjusted by up to 50%. For example, if the splitter comprises two channels with equal cross-sections, the geometric distribution ratio is 1:1. The distribution ratio can be adjusted, for example, to about 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, by using a larger (or smaller) pumping rate than the other fraction to introduce a larger (or smaller) volume of fluid into one fraction. In a preferred embodiment, this geometric distribution ratio ranges from about 2:1 to about 1:2. In other preferred embodiments, this geometric distribution ratio ranges from about 10:1 to about 1:10. The velocity distribution ratio between the upper outlet channel and the lower channel can be approximately 1:10, 1:9.5, 1:9, 1:8.5, 1:8, 1:7.5, 1:7, 1:6.5, 1:6, 1:5.5, 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5:5, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1.

[0222] In one embodiment, the sample fluid containing concentrated particles flows through a fluid channel at a relatively slow rate until it reaches the end of a processing section, where the flow rate increases by at least two times. In a preferred embodiment, the flow rate of the sample fluid increases by at least four times at the end of the processing section. In some cases, the sample fluid is exposed to a magnetic field generated by a magnetic ring or a pair of upper and lower magnets as described herein. In some cases, the sample fluid is not exposed to a magnetic field.

[0223] Aliquots or fractions can be transferred to collection chambers or containers, such as tubes or plates. The fractions or aliquots can then undergo further processing, analysis, or reaction. In embodiments, the collection chambers, plates, wells, and / or tubes include predetermined amounts of desired material or subsequent processing steps, allowing the user not only to concentrate cells but also to transfer them from one medium to another, or to add reagents. Exemplary reagents include, but are not limited to, reagents for RNA isolation, DNA isolation, mRNA isolation, protein isolation, growth media, media, and fixatives. The separated aliquots and / or fractions can be further processed, including magnetic levitation and separation, chemical or biochemical analysis, fractionation, derivatization, sequencing sample preparation, mass spectrometry, NMR analysis, microscopic analysis, FACS sorting and analysis, and X-ray diffraction analysis. Biological cells can be collected in their natural collection state and used for diagnostic or therapeutic procedures, or for genetic or biochemical modifications.

[0224] During their residence in the inlet, processing, or outlet channels, particles can be queried to determine attributes such as velocity, density, biological characteristics, chemical characteristics, genetic characteristics, taxonomic characteristics, configuration, activity, concentration, or orientation. Querying can be performed using one or more sensors or sensor arrays located within, adjacent to, or queriedly connected to the inlet, processing, or outlet channels, or combinations thereof, as well as collection chambers, orifices, plates, or tubes. The detected properties can be used for independent analysis and can be used by system controller components to control or automate system operation. Controlled system components and parameters include sample medium flow rate, magnetic field strength, valve actuation for steering, collection, fractionation, and subsequent reaction conditions. In one embodiment, sensors within the inlet channel are integrated for operative connection to the system controller to provide automated introduction of reagent fluid or gas into the sample stream within the inlet channel.

[0225] Figure 11 The particle concentration using the apparatus of the present invention is illustrated. Particles are introduced into a processing channel through an inlet channel via a sample medium. Sedimentation and / or magnetic repulsion concentrate the particles in the lower layer of the flow stream, and the concentrated particles are collected through a lower outlet channel. A particle removal layer is collected through an upper outlet channel. The method includes the following steps: (i) providing a low-volume fluid apparatus having a processing channel, an inlet channel, and multiple outlet channels; (ii) allowing a particle-containing sample to flow into the processing channel through the inlet channel under certain conditions to generate a sample flow stream having at least a particle enrichment layer and a particle removal layer; (iii) allowing the particle enrichment layer to flow through a first outlet channel to generate a particle enrichment flow stream; (iv) allowing the particle removal layer to flow through a second outlet channel to generate a particle removal flow stream; and (v) collecting one or more flow streams from one or more outlet channels.

[0226] Figure 12 A flow-assisted particle concentration method is illustrated, wherein the inlet channel further comprises a portion substantially linear with the processing channel and a portion nonlinearly aligned with the processing channel, the two portions being connected at an angle θ. A particulate-containing sample medium flows through the inlet channel at a flow rate sufficient to allow sedimentation within the nonlinearly aligned portion of the inlet channel, thereby concentrating the particles within the inlet channel. The particulate-containing sample is kept flowing into the processing channel to provide continuous or further sedimentation, optionally using the magnetic repulsion of the particles to form a particle enrichment layer and a particle removal layer in the flow stream. The layers of the flow stream are collected at their respective outlet channels. Without being bound by any theory, particle aggregation may be the result of the combined effect of two factors: first, the tendency of particles with a density greater than that of the liquid in which they are suspended (e.g., water, solvent) to settle under the presence of gravity; and second, repulsion by a magnetic field. Particle sedimentation can be achieved for at least 1 min, at least 2 min, at least 5 min, at least 10 min, at least 20 min, at least 30 min, or at least 60 min. Particle deposition can be allowed for at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, at least 100 seconds, or at least 200 seconds. In some embodiments, concentrated particles are passed through the fluid channel at a flow rate of up to 50 μL / min, up to 40 μL / min, up to 30 μL / min, up to 20 μL / min, up to 10 μL / min, up to 5 μL / min, or up to 2 μL / min. In some embodiments, concentrated particles are passed through the fluid channel at a flow rate of about 20 μL / min, which will allow liquid to pass through the flow device but allows particles to accumulate outside the magnetic field. In some embodiments, concentrated particles are passed through the fluid channel at a flow rate of about 10 μL / min, which will allow liquid to pass through the flow device but allows particles to accumulate outside the magnetic field. Liquid passing through the flow chamber will deposit through one of the outlet channels leading to the collection chamber. Once most of the liquid has passed through the device and particles are observed entering the flow chamber, the liquid containing suspended particles (e.g., suspended cells) will enter the second output channel at a higher flow rate, which leads to the collection chamber for collecting the target particles.

[0227] Figure 13 The flow-assisted formation of a particle-rich fraction within a processing channel is illustrated. The concentrated (particle-rich) fraction is collected through a lower outlet channel to provide a highly enriched particle aliquot sample. The particle-rich portion of the flow can be identified by sensors (e.g., imaging sensors that observe particles within the processing channel) or temporarily identified via fractions collected at timed intervals, where the graded processing mode within the processing channel has been characterized.

[0228] Figure 14This illustrates pre-concentrating particles within the inlet channel by applying magnetic repulsion before they enter the processing channel. Particle entry into the processing channel can be achieved by: stopping or reducing the magnetic repulsion within the channel; adjusting the flow to overcome the magnetic repulsion; or a combination of magnetic repulsion reduction and flow adjustment. Flow adjustment can include increasing the flow rate of the sample medium or fluctuating the flow, for example, by applying pulsating inlet channel pressure to introduce particles into the processing channel.

[0229] Figure 15 A method for particle concentration is illustrated, wherein a magnetic assembly aligned along the X-axis of a processing channel is provided to extend magnetic repulsion within the inlet channel, thereby preventing particles from entering the processing channel. A flow rate is maintained to provide particle concentration in the inlet channel. After sufficient particle pre-concentration, the magnetic repulsion is overcome, and particles are introduced into the processing channel. The degree of pre-concentration is adjusted by the flow rate of the sample medium. Particles entering the processing channel are deposited / magnetically repelled or attracted into a particle-rich layer of the processing channel flow and collected in the outlet channel.

[0230] According to Figure 16 In one embodiment of the method, air bubbles or immiscible droplets (1601) are introduced after the sample medium enters the processing channel. The air bubbles or droplets flow behind the sample medium in the processing channel to substantially flush away the remaining particle enrichment layer and particle removal layer from the processing channel into the outlet channel. This allows for substantially complete separation of sample fractions and provides processing of discrete samples or sample fractions. The frequency of air introduction can be adjusted to break down large initial sample volumes into smaller units for more efficient processing.

[0231] One embodiment of the method of the present invention is a method for grading blood samples, comprising providing a whole blood sample or a diluted blood sample, subjecting the sample to a sample concentration method as described herein, and separating plasma and / or blood cells from the whole blood or diluted blood sample. The blood sample may be derived from peripheral blood, umbilical cord blood, fetal blood, or arterial blood, wherein the volume is about 50 μL to about 50 mL, 50 μL to about 20 mL, 50 μL to about 10 mL, or about 50 μL to about 5 mL of whole or diluted sample. According to this method, the separated plasma fraction may contain less than about 1% and at least about 0.01% of blood cells in the blood sample, or be substantially free of blood cells. The separated blood sample fraction can be used for diagnostic assays, such as enzyme immunoassays, chemiluminescent immunoassays, hemagglutination / granule agglutination assays, nucleic acid amplification assays, pharmaceutical assays, forensic assays, or genetic trait assays.

[0232] As an example of blood cell concentration / plasma separation, umbilical cord blood samples are fractionated into plasma and cell fractions in the particle concentration apparatus and method of the present invention. A 10 mL blood sample is obtained and a paramagnetic medium of gadobutrol is added to achieve a final concentration of 100 nM. A total sample volume of 500 μL is introduced through the inlet channel of a processing channel configured with a single magnet assembly substantially aligned along the X-axis at the top of the processing channel (dimensions X×Y×Z: 50 × 1.9 × 1 mm), reaches equilibrium in approximately 5 minutes, and then flows at an initial flow rate of 20 μL / min. The combined flow rate of 20 μL / min includes 10 μL / min flowing to the bottom collection channel and 10 μL / min flowing to the top collection channel. After separation is monitored using real-time imaging, the flow rate is increased to 50 μL / min and then to 100 μL / min. The flow rate ratio between the top and bottom collection channels was adjusted from 1:1 to 4:1, and then further adjusted to an asymmetry exceeding 10:1 (with 10 portions entering the bottom collection channel and 1 portion entering the top collection channel) to draw a higher proportion of fluid into the bottom channel, thereby maintaining high purity. Concentration of the blood cell enrichment layer occurs in the bottom layer of the flow stream and is collected through the bottom outlet channel. Figure 16 This is a photomicrograph of the processing channel and sample splitter in the front region (scale bar: flow channel height 1.9 mm), showing the basic concentration and separation of the lower blood cell layer and the upper plasma layer.

[0233] VI. Mobile Room Card Box

[0234] Precision, accuracy, and repeatability are fundamental requirements for scientific instruments. Other considerations include ease of use and manufacturability. The flow cell of this invention possesses all the necessary features of available devices, enabling scientific experiments and research and development work that were previously impossible or could only be accomplished through lengthy, complex, and expensive procedures.

[0235] The flow chamber cartridge of the present invention includes a planar substrate comprising an upper surface and a lower surface, a first longitudinal side surface forming an imaging surface, a second longitudinal side surface forming an illumination surface, and a first and second transverse side surfaces, an inlet orifice on the upper surface, an inlet channel, a sample processing channel fluidly in communication with the inlet channel and positioned substantially parallel to the longitudinal side surface, a sample splitter located within the processing channel, a plurality of outlet channels fluidly in communication with the processing channel, and a plurality of collection orifices fluidly in communication with each of the plurality of outlet channels, wherein the substrate optionally comprises an optically transparent material, and wherein the processing channel is offset in the plane of the substrate to spatially deflect towards the imaging surface. The planar configuration allows all desired flow chamber functions to be integrated into the cartridge and increases performance and reproducibility in laboratory or clinical settings. In operation, it is critical for enhanced performance that the flow through the processing channel and into the outlet channel avoids turbulence as much as possible. The compressibility difference between air and liquid, the influence of channel configuration that can restrict flow, interact with the meniscus of the sample solution, or otherwise induce turbulence will degrade particle separation performance. Minimizing sample flow conditions before the sample enters the processing channel can reduce sample loss, sample adhesion and / or particle agglomeration within the flow chamber channel, and the impact of sample processing on sample cell or organism viability. Features of this invention minimize these and other effects to improve performance and reproducibility. In cases where flow chamber imaging is desired, the planar substrate comprises an optically transparent material. Glass, plastics, or polymeric materials including cyclic olefin polymers (COPs) or cyclic olefin copolymers (COCs) are all suitable for this application. COPs can be used via precision injection molding. Other materials can be used that can form the cartridge by injection molding, etching, laser ablation, machining, or 3D printing. Typical dimensions of the planar substrate can be at least 50 mm long, 20 mm wide, and 1.5 mm thick. Optional ranges include at least 100 mm long, 35 mm wide, and a thickness of about 2 to about 6 mm. The longitudinal sides of the cartridge serve as waveguides for illumination and imaging. Therefore, the processing channel is offset in the plane of the substrate and parallel to and adjacent to the imaging longitudinal sides of the substrate. The distance from the imaging sidewall can be from about 0.5 mm to about 10 mm, preferably from about 0.5 mm to about 5 mm, and optionally from about 1 mm to about 3.5 mm. In an embodiment, the distance between the processing channel and the imaging wall is about 2 mm. The channel size of the processing channel can be any of the embodiments previously described herein. The volume of the processing channel can be configured from about 10 μL to about 800 μL, preferably from about 50 μL to about 600 μL, and optionally from 100 μL to about 400 μL or from about 150 μL to about 300 μL. In some embodiments, the volume is at least about 150 μL, at least about 200 μL, at least about 250 μL, or at least about 300 μL.The combined volume of the outlet channels must be larger than the volume of the processing channels. When operating in a system implementation plan, the flow split between the two outlet channels can be equal, or the ratio can be approximately 4:1 to approximately 1:4, approximately 3:1 to approximately 1:3, or approximately 2:1 to approximately 1:2, or it can be based on 1:1 with a fluctuation of approximately 50% or less, or approximately 40% or less, or approximately 30% or less, or approximately 15% or less.

[0236] The flow chamber of the present invention optionally includes a collection orifice located on a planar substrate. The collection orifice is characterized by an inlet in fluid communication with an outlet channel, wherein the inlet is at a first orifice height and is configured with a step transitioning from the inlet orifice to the bottom plate of the orifice. This provides a transition surface for sample fractions to flow into the orifice and can suppress reverse siphoning of sample fractions into the outlet channel and bubble formation within the collection orifice. The outlet channel within the collection orifice is provided with an opening at a height above the opening of the inlet channel from the bottom plate of the collection orifice. An internal outlet tank is positioned in communication with a flow regulator, in some instances, a separate pump to provide flow through the flow chamber. In operation, the collection orifice is sealed with a layer of material or a membrane to provide a closed system, thereby allowing the sample and sample fractions to flow or be pumped through the flow chamber. It is important to provide a biocompatible adhesive when assembling the flow chamber layers and when using adhesives. Proper selection of the adhesive is essential to minimize or prevent adhesive components from leaching into the solution, adhering to cells or binding molecules from the solution, generating autofluorescence, having textures that increase surface area and thus affect cells, and being excessively hydrophilic or hydrophobic. Preferred adhesives are silicone or silicone-based adhesives.

[0237] VII. Cell Separation System

[0238] The cell separation system of the present invention includes a receiving block for holding a flow chamber cartridge, an optical system including an optical sensor, a lens, and an illumination source, and a plurality of flow conditioning components. The receiving block removably positions the flow chamber cartridge in optical alignment with the optical system, removably engages a magnetic component adjacent to a processing channel of the flow chamber, and removably positions a plurality of outlet channels of the flow chamber cartridge in fluid communication with the plurality of flow conditioning components. The optics are configured to provide microscopic imaging of the processing channel of the flow chamber cartridge. Optionally, the optical system is configured and arranged to provide imaging for fluorescence emission using an optional ultraviolet exciter module. The optical system may include a visible light illumination source configured and arranged to provide light transmission through the processing channel within a planar substrate. The receiving block is configured and arranged to hold the planar flow chamber cartridge in an orientation relative to the optical system such that the imaging optics are aligned with the imaging side of the planar cartridge, and the visible light emitter is positioned in an orientation illuminating the illumination side of the planar flow chamber cartridge. Optionally, the optical system may also include one or more ultraviolet illumination sources configured and arranged such that the ultraviolet illumination sources, optionally at wavelengths of about 474 nm and / or 560 nm, are placed at an angle on the imaging side of the planar cell to excite fluorophores within the processing channel of the cell.

[0239] To image the fluorescent entity inside the processing channel, the optical system may optionally include a dual bandpass filter, which preferably allows emitted radiation in the bands centered at wavelengths of about 524 nm and 628 nm to pass through.

[0240] An optional feature of the receiving block is a series of flow regulator adapters that interface with outlets on the top or bottom of the flow chamber cartridge. The adapters facilitate fluid communication with a flow regulator (e.g., a pump in the system) and with the outlet channels of the flow chamber (e.g., collection orifice outlet channels). Once the flow chamber cartridge is inserted into the receiving block, the receiving block is mechanically actuated to support the cartridge, align the illumination and imaging sides of the flat cartridge with the optical imaging system, align the magnetic components to their positions above and below the flow chamber processing channels, and, where desired, position the flow regulator adapters in fluid communication with the corresponding outlet channels of the flow chamber cartridge.

[0241] The system's flow regulator provides flow rates to the sample and sample fractions in the flow chamber cartridge. During separation, the flow rate provided by the flow regulator can range from as low as 1 μL / min to as high as 1 mL / min. Flow rates can be approximately 25 μL / min, approximately 50 μL / min, approximately 100 μL / min, approximately 200 μL / min, approximately 250 μL / min, approximately 300 μL / min, or approximately 300 μL / min to approximately 1 mL / min. The total sample volume flow rate can be approximately 50 μL / min, approximately 75 μL / min, approximately 100 μL / min, approximately 150 μL / min, approximately 200 μL / min, or approximately 300 μL / min. When operating in a system implementation plan, the flow split between the two exit channels can be equal, or the range can be approximately 4:1 to approximately 1:4, approximately 3:1 to approximately 1:3, or approximately 2:1 to approximately 1:2, or it can be based on 1:1 and fluctuate by approximately 50% or less, or approximately 40% or less, or approximately 30% or less, or approximately 15% or less.

[0242] The magnetic components of the system may include the materials, dimensions, and strengths described above, and may be placed in the configurations described above and below.

[0243] VIII. Separation and Isolation of Cells and Nuclei

[0244] In a first aspect, this disclosure provides a method (“Method 1”) for extracting cell nuclei from intact cells, comprising: providing intact cells; and lysing the cells in a nucleus separation buffer; wherein the nucleus separation buffer contains wheat germ lectin (WGA). Wheat germ lectin (WGA) is a lectin that protects wheat from insects, yeast, and bacteria. It is capable of masking glycosylated moieties, such as N-acetyl-D-glucosamine and sialic acid, and is commonly used in a fluorescently labeled state to label cell nuclei, and is routinely used for staining bone and myocardial membranes to determine cross-sectional area or muscle cell density.

[0245] According to the present invention, it has been found that adding WGA to preparations for isolating cell nuclei from cells and / or to preparations for isolating RNA from isolated cell nuclei can result in a higher RNA yield from the isolated cell nuclei.

[0246] Figure 30Results of large-scale RNA extraction with and without WGA are shown. Data on total RNA yield in nuclear separation buffer with and without WGA, as well as ng RNA / nucleus data for resuspended nuclei in separation buffer with and without WGA, are provided. It can be seen that the addition of WGA, particularly at 0.1 mg / mL, yields more RNA than separation buffer alone.

[0247] The nucleus separation buffer can be any known buffer system containing detergent for separating nuclei from cells. Typically, the concentration of WGA is from about 0.01 mg / mL to about 2 mg / mL; for example, from about 0.01 mg / mL to about 1 mg / mL; for example, from about 0.01 mg / mL to about 0.5 mg / mL; for example, from 0.05 mg / mL to about 0.15 mg / mL; for example, about 0.1 mg / mL.

[0248] In other embodiments, the method further includes, for example, collecting cell nuclei by centrifugation. The cell nuclei can then be stored, for example, by suspending them in a storage buffer; wherein the storage buffer contains, for example, sucrose. In other embodiments, the integrity of the isolated cell nuclei is at least about 15% higher than that of cell nuclei isolated by a similar method lacking WGA. In other embodiments, the method further includes suspending the collected cell nuclei in a suspension buffer containing a suspending agent. In other embodiments, the suspending agent contains 100 mM gadolinium, and the suspension buffer contains 1X PBS, 1% BSA, and an RNAse inhibitor, such as RNAseOUT. TM In other embodiments, the method further includes using the isolated cell nucleus to perform any of the methods described herein, including method 2 and subsequent methods.

[0249] Secondly, as has been found in this disclosure, particles present in the sample and / or flow stream of the apparatus described herein can influence the movement of matter in the sedimentation direction in a size-dependent manner. The particles referred to herein as "separation particles" can be any microbead-like particles used for chromatographic separation and may optionally be coated with proteins, for example. Examples of separation particles include agarose microbeads such as Sepharose. TM Microbeads, dextran microbeads such as Sephadex TMPolyacrylamide microbeads, glucose microbeads, polystyrene microbeads, microbeads made from polymeric resins such as polyvinylethyl carbitol, polyvinylpyrrolidone, cellulose, and silicone-based materials, and / or microbeads made from mixtures such as dextran-polyacrylamide, such as Sephacryl™ microbeads; each type of microbead may optionally be coated, for example, with a protein. In a specific embodiment, the separated particles are polystyrene microbeads or particles; for example, streptavidin-coated polystyrene microbeads or particles, such as SPHERO microbeads sold by Spherotech Corporation (Lake Forest, Illinois). TM Streptavidin-coated particles.

[0250] Method 2 and subsequent methods of this invention provide positive or negative selection strategies for purifying cell and / or particle nuclei as described herein. While not wishing to be bound by a particular theory, it is thought that separating particles can a) form complexes or bind non-nuclear components of the sample mixture (i.e., contaminants, such as dead cells, cell debris, nuclear aggregates, and / or other components); and / or b) form a matrix or barrier within the flow stream that traps or prevents the free movement of contaminants; either of these can influence the movement of matter in the sedimentation direction in a size-dependent manner. For example, it has been observed that the presence of 3-micron streptavidin-coated polystyrene beads in the sample and / or flow stream, which suspend at higher band positions in the flow stream, creates a negative selection mechanism in which non-nuclear components of the sample mixture are nonselectively retained in the streptavidin-coated polystyrene beads in the higher bands of the flow stream, while individual nuclei filter through (i.e., precipitate through) the streptavidin-coated polystyrene beads and can be collected at lower levels in the flow. See Example 5 below.

[0251] It should be understood that the method of the present invention also facilitates the separation of target subcellular components that are more buoyant (and / or denser) than the separating particles. For example, it is conceivable that selecting separating particles with lower buoyancy will cause the separating particles to settle in the lower band of the flow channel, while target subcellular components with higher buoyancy than the separating particles will remain in the higher band of the flow, thus allowing the components in the higher bands to be collected. Therefore, by selecting separating particles with appropriate buoyancy and / or density in relation to the buoyancy and / or density of the desired target subcellular components, the separation of the desired target subcellular components can be facilitated.

[0252] A method for separating a mixture of live and dead cells includes: providing a flow chamber cartridge, such as the flow chamber cartridge of the above embodiment, the flow chamber cartridge comprising a processing channel and a plurality of outlet channels, wherein the combined volume of the outlet channels of the flow chamber cartridge is greater than the volume of the processing channel; allowing a sample solution containing live and dead cells, along with a paramagnetic compound, to flow into the processing channel; placing the flow chamber cartridge in a magnetic field substantially parallel to and aligned with the processing channel; completely holding the processing channel and the sample contained therein in a stopped-flow state within the magnetic field for a sufficient time to allow the live and dead cells to separate at a vertical distance within the processing channel; simultaneously discharging a sample fraction rich in live cells and a sample fraction rich in dead cells into the outlet channels; optionally, the method further includes providing a flow chamber cartridge substantially free of any liquid or paramagnetic compound prior to the introduction of the sample solution.

[0253] The flow chamber cartridge used in the method of the present invention may include an outlet channel with a cross-sectional area smaller than that of the processing channel, and is arranged in a compact path arrangement; an exemplary configuration is a serpentine channel. Magnetic fields are placed immediately adjacent to the top vertical surface and immediately adjacent to the bottom vertical surface of the processing channel, each magnetic field having a similar strength and a surface field strength between about 0.5 Tesla and about 2.0 Tesla, and optionally between about 0.9 Tesla and about 1 Tesla. The surface field strength may be about 0.5 Tesla, about 0.6 Tesla, about 0.7 Tesla, about 0.8 Tesla, about 0.9 Tesla, or about 1.0 Tesla.

[0254] The method further includes providing a paramagnetic compound in the sample solution at a concentration of about 50 mM to about 200 mM, optionally about 65 mM to about 175 mM, and further optionally about 70 mM to about 150 mM. The concentration may be about 70 mM, about 75 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM.

[0255] The method may also include the step of discharging sample fractions into an outlet channel at a flow rate of about 75 uL / min to about 150 uL / min, and optionally about 75 uL / min, about 90 uL / min, about 100 uL / min, about 110 uL / min, about 120 uL / min or about 150 uL / min.

[0256] This method produces extremely high recovery rates and purity of live cell fractions. The enriched recovered sample fractions contain at least about 60%, at least about 70%, at least about 80%, or at least about 90% live cells, and the yield of live cells in the enriched recovered sample fractions is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the sample.

[0257] Experiments were conducted on different instruments with varying flow chamber configurations to separate live and dead Jurkat cells. A population of dead Jurkat cells was generated by treating fresh Jurkat cells with 70% ethanol. After removing the ethanol and washing the dead cells in the medium, a mixed population was generated by mixing the dead cells back into the original live cell population. The concentration of dead cells in this final mixture was approximately 20%. Aliquots of the cell mixture were separated using flow chambers and systems as described herein, under conditions optimized for each instrument configuration. Figure 25A and 25B The viability and yield of live cell fractions obtained by the method described herein are shown. Figure 2 A and 26B demonstrate that the methods described herein enable the efficient isolation of live cells of various cell types. Live cell isolation and enrichment have been performed on primary cells, primary cells isolated from human and animal tissues, dissociated cells from tumors, cultured cells, and other cells described herein.

[0258] The separation of cell nuclei or particles is achieved rapidly, without subjecting the nuclei to stress associated with other washing and / or separation techniques such as FACS or wash / centrifugation. Rapid separation of cell nuclei is performed by a method comprising: loading a sample containing cell nuclei and a sample medium containing a paramagnetic compound or ferrofluid into a separation channel; subjecting the sample to magnetic force using at least one magnet to achieve separation; collecting at least one fraction of the separated sample containing cell nuclei without further centrifugation; and optionally imaging the particles in the sample before, during, and / or after separation. In this method, the sample may contain approximately 50 to approximately 10,000,000 cell nuclei. The total separation time can range from approximately 1 minute to approximately 20 minutes. In rapid separation methods, the concentration of target cell nuclei increases in solution or a portion of solution at a ratio of at least about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1.5:1, or 1.1:1. Cell nuclei in a solution containing multiple particle types can be considered “separated” if, after solution treatment, the ratio of target cell nuclei to other particle types increases, or if the ratio increases by at least about 10%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, or if the concentration of non-nuclear particles in solution decreases by at least about 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%. In a preferred embodiment of the cell nucleus enrichment method, the integrity of the isolated cell nuclei in the enriched collected sample portion is 30% higher than the integrity of similar cell nuclei isolated by methods including centrifugation. Preferably, the integrity of the isolated cell nuclei in the enriched collected sample portion is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the integrity of similar cell nuclei isolated by methods including centrifugation. Integrity refers to the integrity of the nuclear membrane, which results in a substantial proportion of DNA and RNA being contained within the isolated cell nuclei.The starting sample may contain about 50 to about 10,000,000 cell nuclei, about 500 to about 10,000,000 cell nuclei, about 2,000 to about 10,000,000, about 10,000 to about 10,000,000, about 25,000 to about 10,000,000, about 50,000 to about 10,000,000, about 75,000 to about 10,000,000, about 100,000 to about 10,000,000, about 150,000 to about 10,000,000, about 200,000 to about 100,000,000, or about 500,000 to about 10,000,000 particles. The time for separating and collecting the separated particle fractions can be 20 minutes or less, 18 minutes or less, 15 minutes or less, 12 minutes or less, 10 minutes or less, 8 minutes or less, 5 minutes or less, 3 minutes or less, or less than 1 minute. The cell nuclei separated by the above methods can include human cells, non-human animal cells, plant cells, and eukaryotic cells (e.g., but not limited to immune cells, endothelial cells, yeast, and T cells). The separated cell nuclei can be mononuclear, binuclear, or multinucleated from enucleated cells. Various cell types can include dead cells, live cells, healthy cells, pathological cells, infected cells, transfected cells, or genetically modified cells. The cell nuclei separated according to the methods of this disclosure can be obtained directly from an organism or from proliferating or cultured cells. In a specific embodiment of the rapid, high-volume separation method, live cells are separated from dead cells. In another embodiment of the separation method, cell nuclei are separated from live transfected cells and / or CRISPR-modified or otherwise gene-edited cells. In another embodiment of the method, cell nuclei are separated from cells, cell debris, and / or sample debris. In various implementations, these methods utilize the boxes and systems described herein.

[0259] Example 1: Isolation of cell nuclei from rapidly frozen lung and brain tissue

[0260] Nuclei were isolated from rapidly frozen lung and brain tissue by incubation on ice with nuclear lysis buffer (ThermoFisher) for 30 minutes. The nuclei were then washed twice with PBS containing 0.1% BSA and resuspended in nuclear storage buffer (containing 1 M sucrose). According to the invention, the use of sucrose in the nuclear storage buffer has been found to promote the storage of nuclei. Nuclei for isolation were prepared by resuspending 3e⁵ nuclei in suspension buffer containing 100 mM suspending agent. After isolation, one sample was retained for comparison. Nuclei were isolated using a 30-minute equilibration time in the separator device described herein (LeviCell, LevitsBio, Menlo Park, CA). The output was stained with PI and imaged using an Echo microscope.

[0261] Cell nuclei stain brightly with PI, while cell debris does not. LeviCell's staining input and output images showed a significant reduction in the amount of unstained debris in both tissue samples. Lung tissue produced more debris than brain tissue. Figure 27 (A and C), but after sorting with LeviCell, both samples showed a significant reduction in fragments ( Figure 27 (B and D).

[0262] Unsorted (from Jurkat cells) Figure 28 A) and sorting ( Figure 28 B) Flow cytometry analysis of cell nuclei. Samples were stained with PI and CellBrite Fix Green for 15 minutes at room temperature and then analyzed on a Sony SH800S sorter. After sorting, the populations of debris and dead cells were reduced, and the nuclear population was enriched (50.81% to 63.57%). In addition, the population of single nuclei increased from 28.28% to 43.98%.

[0263] Example 2 - Isolation of cell nuclei from Jurkat cells

[0264] Jurkat cells (ATCC# TIB-152) were aged for several days without altering the culture medium to induce cellular senescence and apoptosis. These cells were collected and counted using an AO / PI analyzer on a Nexcelom spectrometer. A portion of these cells was retained as a total RNA input control. Nuclei of approximately 50% viability were extracted from 8 million cells using nuclear EZ lysis buffer (Sigma) containing the RNAse Out inhibitor (Thermo). The nuclei were precipitated and resuspended in a suspension buffer containing 100 mM gadolinium, 1X PBS, 1% BSA, and 0.2 U / μl RNAse Out—this sample is now referred to as the input sample. 1 million extracted nuclei were suspended in a LeviCell system for 30 minutes. Nuclei were collected from the bottom channel of the LeviCell cartridge and, parallel to the nuclei extracted in the previous step (input), whole cells were retained from the initial sample and treated for total RNA using the RNEasy Prep Mini Kit (Qiagen). Total RNA was normalized, and 10 ng of total RNA was used to prepare cDNA using the PrimescriptRT with gDNA eraser kit (Takara). A pre-designed and validated PrimeTime qPCR probe assay (IDT) targeting 18S, SCARNA5, and SNHG6 was performed in a 10 μl reaction volume containing 5 μl of 2x Primetime gene expression premix (IDT) and 200 pg equivalent cDNA. All reactions were performed in triplicate on a Quanttsudio5 (Thermo) instrument. Amplification data were analyzed using the ΔΔCT method and reported as relative amounts normalized to the 18S endogenous control, compared with whole-cell RNA.

[0265] Result: As Figure 29 As shown, nuclei processed on the LeviCell system exhibited higher enrichment for nuclear-specific targets (SCARNA5) and less contaminating cytoplasmic targets (SNHG6) compared to input nuclei. Nuclei processed on the LeviCell system showed almost 3-fold enrichment of nuclear-specific signals compared to intact cells, but showed no enrichment of cytoplasmic targets. Suspension samples also showed 50% higher enrichment of nuclear-specific signals compared to input nuclei. In contrast, input nuclei showed less enrichment of nuclear-dependent target signals and a greater amount of contaminating cytoplasmic RNA compared to whole-cell RNA. These data suggest that nucleus suspension on the LeviCell system enables the enrichment of both nuclear and nuclear-specific RNA compared to nuclei extracted using conventional methods.

[0266] Example 3 - Effect of WGA on the Yield of Isolated Nuclear RNA

[0267] Nuclei were isolated from human brain cells by incubation in nucleus separation buffer containing 0.1 mg / mL WGA or 0.3 mg / mL WGA. The resulting nuclei were resuspended in the same buffer, and the RNA content in each preparation was determined. Figure 30 Data on total RNA yield in nuclear separation buffers with and without WGA, as well as ng RNA / nucleus data for resuspended nuclei in separation buffers with and without WGA, are presented. It can be seen that the addition of WGA, particularly at 0.1 mg / mL, results in an increased RNA yield when performing large-volume RNA extractions.

[0268] Example 4 - Separation of human brain cell nuclei using LeviCell with 3-micron streptavidin-coated polystyrene microbeads

[0269] LeviCell was used to isolate human brain cell nuclei from preparations containing cell nuclei and large and small cell debris. Human brain cell nuclei were isolated and pre-stained with propidium iodide in wash buffer, then resuspended in PBS / BSA / propidium iodide. Then, 3–3.4 μm streptavidin-coated polystyrene beads (SVP-30-5, Spherotech, LakeForest IL) were added to a concentration of approximately 130 beads / nucleus. A control without beads was also prepared. Gadolinium suspension buffer was added to the sample to provide 150 mM gadolinium, and the sample was loaded onto the LeviCell.

[0270] The results are shown in Figure 31 The image shown is an image of the flow chamber during separation. It can be seen that the two samples containing microbeads (“(+) microbeads”) have dense bands in the upper lane consisting of microbeads and mostly non-nuclear debris. Conversely, the two control samples without microbeads (“(-) microbeads”) show significantly less nuclear separation. Figure 32 Images from the top and bottom fractions of the LeviCell cell counting system show that the nuclei obtained in the presence of microbeads (bottom right) appear to be rounder, in better condition, and mostly single nuclei. Conversely, cells obtained without microbeads (top right) appear to contain more aggregates (multiples) and abnormal (elongated) nuclei.

[0271] These results suggest that the microbeads appear to be capturing nuclear aggregates and potentially membrane fragments, and that the presence of microbeads during separation improves the appearance of the cell nucleus.

[0272] Example 5 - Using 3-micron streptavidin-coated polystyrene microbeads, human brain cell nuclei were isolated and RNA sequenced using LeviCell.

[0273] As described above, human brain cell nuclei were isolated using LeviCell and then their RNA was sequenced.

[0274] Isolate cell nuclei from human brain tissue according to the following steps. Mince the tissue and homogenize it in nucleus separation buffers containing and without WGA. Filter the sample through a 70 μm molecular sieve with PBS / BSA / RNAseOUT, wash (centrifuge) with 1x PBS / BSA / RNAseOUT, and resuspend in PBS / BSA / RNAseOUT.

[0275] For Group 1, two libraries were prepared: 250 μl each of 1 (5rxn equivalent w / PI) and 1 (5rxn equivalent NO Pi). 375 μl of PBS / BSA / PI or PBS / BSA / rnase inhibitor was added to each library to a final volume of 625 μl. Each library was aliquoted into 125 μl portions, and 80 μl of microbeads (approximately 165 microbeads / nucleus) was added to each portion and incubated at room temperature for 5 minutes, or 80 μl of PBS was added as a control. 70 μl of suspension buffer was added to achieve a final concentration of 150 mM Gd. The sample was loaded onto Levicells at a concentration of 150 mM Gd 1% Pi, and nuclear yield and purity of the bottom fraction were measured.

[0276] For group 2, 75 μL of PBS / BSA / rnase inhibitor was added to 50 μL of each preparation, followed by incubation at room temperature with 80 μL of microbeads (approximately 100-130 microbeads / nucleus), or 80 μL of PBS was added as a control. 70 μL of suspension buffer was added to achieve a final concentration of 150 mM Gd. The samples were loaded onto Levicells at a concentration of 150 mM Gd 1% Pi, and nuclear yield and purity of the bottom fraction were measured. Parallel experiments without propidium iodide were also performed for subsequent RNA sequencing using the 3' RNA LT kit, targeting approximately 700 nuclei / sample.

[0277] result: The bottom fraction yields and purities of the samples are shown in the table below:

[0278] It can be seen that sample 15, which contains microbeads and cell nuclei, used WGA in the preparation, has high yield and high purity.

[0279] Figure 33Spectral data for pre-stained cell nuclei with and without microbeads are shown (preparations R7 and R8). The input spectrum of the microbead-containing sample (top right panel) shows dense debris areas in regions of lower cell intensity compared to the non-microbead sample (top left panel), which is partly due to the presence of microbeads. The average fluorescent particle size for the input microbead-containing sample (R8) is approximately 10 micrometers. For the cell nuclei (the band at the top of the panel), this value decreases to approximately 8 micrometers in the output spectrum (bottom right panel), with a significant reduction in debris. This effect was not observed in the non-microbead sample.

[0280] RNA sequencing results: RNA in the sample was sequenced using a 10×3' RNA LT kit, targeting approximately 700 cell nuclei per sample. Figure 34 The Seurat rearrangement diagram of the sequencing samples is shown. As can be seen from the mitochondrial percentage chart, samples 7 and 8, which contain both WGA and microbeads, have fewer mitochondrial readings compared to any of the other experiments. Less ribosomal RNA was also present in these preparations, and ribosomal RNA is a cytoplasmic contaminant in nuclear preparations.

[0281] IX. The disclosed implementation schemes are non-restrictive.

[0282] While various embodiments of the invention have been shown and described herein, it is important to emphasize that these embodiments are provided by way of example only. Many variations, alterations, and substitutions may be made in these various embodiments without departing from the invention. Specifically, when any scope is described herein, unless otherwise expressly stated, the scope includes all values ​​therein and all sub-scopes therein.

[0283] Furthermore, and more generally, conventionally applied fluid dynamics, molecular biology, cell biology, microbiology, and recombinant DNA technologies within the scope of this art can be employed based on the disclosures, discussions, embodiments, and implementations herein. Resources incorporated herein by reference are for their respective contents and the teachings found therein. Such inclusions are at least for specific teachings and / or other purposes that may be indicated when citing references herein. Where no specific teachings and / or other purposes are indicated, published resources are specifically incorporated for the teachings indicated by one or more of the titles, abstracts, and / or summaries of the references. Where no specifically identified teachings and / or other purposes may be so relevant, published resources are incorporated to more fully describe the prior art to which this invention relates, and / or to provide teachings that may be applicable and are generally known to those skilled in the art. However, it is specifically noted that references to disclosed resources herein should not be construed as an admission that they are prior art to this invention. Furthermore, where one or more of the incorporated published resources differ from or contradict this application, including but not limited to defined terminology, terminology usage, described techniques, etc., this application is considered the preferred embodiment, and any contradiction may be regarded as an alternative embodiment.

[0284] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these preferred embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention.

Claims

1. A method for extracting a cell nucleus from an intact cell, comprising: Provide complete cells; as well as The cells were lysed in a cell nucleus separation buffer; The cell nucleus separation buffer contains wheat germ lectin (WGA).

2. The method of claim 1, wherein the WGA is present in an amount of about 0.01 mg / mL to about 2 mg / mL; for example, about 0.01 mg / mL to about 1 mg / mL; for example, about 0.01 mg / mL to about 0.5 mg / mL; for example, 0.05 mg / mL to about 0.15 mg / mL; for example, about 0.1 mg / mL.

3. The method according to claim 1 or claim 2, wherein the nuclear separation buffer comprises the WGA and one or more buffers, solvents, proteins, particles, enzymes and stabilizers; and water.

4. The method according to any one of claims 1-3 further comprises: Collect cell nuclei, for example, by centrifugation; as well as The cell nucleus may be stored optionally; For example, by suspending the cell nucleus in a storage buffer; For example, the storage buffer described therein contains sucrose.

5. The method of claim 1, wherein the integrity of the isolated cell nuclei is at least about 15% higher than that of cell nuclei isolated by a similar method lacking WGA.

6. The method according to any one of claims 1-5 further includes suspending the collected cell nuclei in a suspension buffer containing a suspending agent.

7. The method of claim 6, wherein the suspending agent comprises 100 mM gadolinium, and the suspension buffer comprises 1X PBS, 1% BSA, and an RNAse inhibitor, such as RNAseOUT. TM .

8. A method for separating a target subcellular component, such as a cell nucleus, from a sample comprising a target subcellular component and one or more contaminants, comprising: A sample comprising the target subcellular components, contaminants, and sample medium is loaded into a separation channel, optionally causing the sample to flow along the separation channel, wherein the sample medium comprises: i) Paramagnetic compounds or ferromagnetic fluids; and ii) Separate particles (or microbeads); At least one magnet is used to apply a magnetic force to the sample to achieve the separation of the target subcellular component from other components of the sample; Collect at least one fraction of the isolated sample containing the target subcellular components without further centrifugation; as well as Optionally, the target subcellular components in the sample can be imaged before, during, and / or after separation; The size of the separated particles is about 1 to about 15 micrometers; and wherein: a) The separated particles form a complex with one or more contaminants, or the separated particles form a structure in the sample medium that interacts with one or more contaminants by inhibiting the movement of the one or more contaminants in a selected direction relative to the target subcellular component; or b) The separated particles form a complex with the one or more contaminants, or the separated particles form a structure in the sample medium that interacts with the one or more contaminants by increasing the movement of the one or more contaminants in a selected direction relative to the target subcellular component.

9. The method of claim 8, wherein the particles form a complex with one or more pollutants, or form a structure in the sample medium that interacts with one or more pollutants, in a manner that inhibits the movement of the one or more pollutants in the direction of environmental gravity relative to the target subcellular components.

10. The method according to claim 8 or claim 9, wherein the target subcellular component is the cell nucleus.

11. The method of claim 10, wherein the contaminant comprises one or more of intact cells, dead cells, nuclear fragments, and cell fragments.

12. The method according to any one of claims 7-11, wherein the size of the separated particles is from about 1 to about 10 micrometers; for example, the size is from about 1 to about 8 micrometers; for example, the size is from about 1 to about 5 micrometers; for example, the size is from about 1, about 2, about 3, about 4 or about 5 micrometers.

13. The method according to any one of claims 7-12, wherein the separated particles are selected from agarose microbeads such as Sepharose. TM Microbeads, dextran microbeads such as Sephadex TM Polyacrylamide microbeads, glucose microbeads, polystyrene microbeads, microbeads made of polymer resins such as polyvinyl ethyl carbitol, polyvinylpyrrolidone, cellulose, and silicon-based materials, and / or microbeads made of mixtures such as dextran-polyacrylamide such as Sephacryl™ microbeads; each of these microbeads may optionally be coated, for example, with proteins; for example, polystyrene microbeads or particles coated with streptavidin.

14. The method according to any one of claims 7-13, wherein the separated particles are optionally coated polystyrene particles; for example, polystyrene particles coated with protein.

15. The method of claim 14, wherein the separated particles are polystyrene particles coated with streptavidin.

16. The method of claim 15, wherein the size of the separated particles is about 3 micrometers.

17. The method of claim 8, wherein the sample comprises about 50 to about 10,000,000 cell nuclei.

18. The method of claim 8, wherein the concentration of cell nuclei in the fraction is increased by at least 1.1:1 compared to the original sample.

19. The method of claim 8, wherein the concentration of non-nuclear particles in the original sample is reduced by at least about 1% in the fraction.

20. The method of claim 8, wherein the integrity of the isolated cell nuclei from the fraction of the sample is at least about 30% higher than the integrity of cell nuclei isolated from the fraction of the sample by means including centrifugation.

21. The method according to claim 8, wherein the cell nucleus is isolated from human cells, non-human animal cells, or plant cells.

22. The method according to claim 1, wherein the cell nucleus is isolated from healthy cells, diseased cells, infected cells, transfected cells, or genetically modified cells.

23. A method for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells and cell debris, said method comprising: A) Provides a fluid sample processing apparatus, the fluid sample processing apparatus comprising... (i) Processing channels; (ii) Entrance passage; (iii) An inlet connection area that connects the inlet channel to the processing channel; (iv) Multiple magnetic components aligned along the X-axis of the processing channel on the upper and lower sides of the processing channel; (v) Multiple exit channels; (vi) Export connection area, which connects the processing channel to the export channel; (vii) A first outlet channel, which is in fluid communication with the upper region of the processing channel at the outlet connection area; (viii) A second outlet channel, which is in fluid communication with the lower region of the processing channel at the outlet connection area; and (ix) A first flow regulator associated with the first outlet channel and a second flow regulator associated with the second outlet channel; as well as B) Flow the mixture through the fluid sample processing device to provide a first recovered sample rich in the cell nuclei and / or live cells; and a second recovered sample from which the cell nuclei and / or live cells have been removed; The mixture comprises: i) paramagnetic compounds or ferrofluids; and ii) Separate particles; The size of the separated particles is about 1 to about 15 micrometers; and wherein: a) The separated particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, by inhibiting the movement of at least a portion of the cell debris in a selected direction relative to the cell nucleus; or b) The particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

24. The method of claim 23, wherein the particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, in a manner that inhibits the movement of at least a portion of the cell debris in the direction of environmental gravity relative to the cell nucleus.

25. The method of claim 24, wherein the first recovered sample is rich in cell nuclei.

26. The method of claim 24, wherein the first recovered sample is rich in live cells.

27. The method according to claim 23, wherein: a) The yield of live cells in the first recovered sample is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total live cell composition of the mixture; and / or b) The yield of cell nuclei in the first recovered sample is at least about 50%, at least about 60%, at least about 70%, or at least about 75% of the total cell nuclei derived from the mixture of living cells.

28. The method of claim 23, wherein the outlet connection area further comprises a flow splitter portion.

29. The method of claim 28, wherein the flow splitter portion is advanced into the processing channel and is configured and arranged to separate the fluid flow into separate flows in the outlet channel.

30. The method according to any one of claims 23-29, wherein the fluid sample processing apparatus further comprises a first flow rate sensor associated with a first outlet channel and a second flow rate sensor associated with a second outlet channel.

31. The method of claim 30, wherein the flow rate sensor is operatively connected to the flow regulator.

32. The method of claim 23, wherein the fluid sample processing apparatus further comprises an optical sensor and an illumination source, the illumination source being configured to be opposite or angularly adjacent to the optical sensor; optionally, wherein the illumination source emits ultraviolet light.

33. The method of claim 23, comprising: The sensor is a photodetector, a multi-pixel imaging detector, a magnetic field detector, an electrochemical detector, an optical phase detector, a scattering detector, a Hall sensor, a magnetoresistive sensor, a radiation thermal sensor, a surface acoustic wave sensor, a biosensor, a capacitive sensor, a conductive sensor, a thermal sensor, a flow rate sensor, an ultrasonic sensor, a gravity sensor, a magnetic field sensor, or a combination thereof. as well as A controller that can be operatively connected to multiple flow regulators.

34. The method of claim 23, wherein the fluid sample processing apparatus comprises a flow chamber cartridge containing a planar substrate, the planar substrate comprising: (i) Upper and lower surfaces; (ii) Forming the first longitudinal side surface of the imaging surface; (iii) Forming a second longitudinal side surface of the illumination surface; as well as (iv) First transverse side and second transverse side; (v) The inlet hole located on the upper surface; (vi) Entrance passage; (vii) Sample processing channel, which is in fluid communication with the inlet channel and is positioned substantially parallel to the longitudinal side; (viii) Sample splitter, located within the processing channel; (ix) Multiple outlet channels, which are in fluid communication with the processing channels; as well as (x) Multiple collection holes, which are in fluid communication with each of the multiple outlet channels; The substrate may optionally comprise an optically transparent material, and the processing channel is offset in the plane of the substrate to spatially deflect towards the imaging surface. Optionally, the substrate comprises a non-ferrous metal, ceramic, glass, polymer, or plastic; and Optionally, if the substrate comprises one or more layers, the substrate and the planar layer may comprise the same or different materials.

35. The method of claim 23, wherein the fluid sample processing apparatus comprises a flow chamber cartridge containing a planar substrate, the planar substrate comprising: (i) An inlet hole located on the upper surface; (ii) Entrance passage; (iii) Sample processing channel; (iv) Sample splitter, located within the processing channel; (v) Multiple outlet channels that are in fluid communication with the processing channels; as well as (vi) Multiple collection holes, which are in fluid communication with each of the multiple outlet channels; The substrate comprises an optically transparent material, and the combined volume of each of the plurality of outlet channels is greater than the volume of the processing channel; Optionally, the substrate comprises a non-ferrous metal, ceramic, glass, polymer, or plastic; and Optionally, if the substrate comprises one or more layers, the substrate and the planar layer may comprise the same or different materials.

36. The method of claim 34, wherein the outlet channel of the flow chamber cartridge is arranged in a compact path, for example, wherein the outlet channel is a serpentine channel.

37. The method of claim 35, wherein the outlet channel of the flow chamber cartridge is arranged in a compact path, for example, wherein the outlet channel is a serpentine channel.

38. The method according to claim 36, wherein: The outlet channel of the flow chamber cartridge is formed as a recess within a planar substrate, and a first outlet channel includes a recess on the surface of the planar substrate, and a second outlet channel includes a recess on an opposite side of the planar substrate; optionally, the channels are formed by etching, machining, 3D printing, or molding the planar substrate; and / or The flow chamber also includes one or more additional planar layers positioned above the recess in the planar substrate to form a closed channel; optionally, the one or more planar layers are attached to the planar substrate by pressing, adhesive bonding (preferably with a biocompatible adhesive, more preferably with silicone or silicone-based adhesives), solvent bonding, ultrasonic welding, thermal bonding, welding, or 3D printing.

39. The method of claim 37, wherein: The outlet channel of the flow chamber cartridge is formed as a recess within a planar substrate, and a first outlet channel includes a recess on the surface of the planar substrate, and a second outlet channel includes a recess on an opposite side of the planar substrate; optionally, the channels are formed by etching, machining, 3D printing, or molding the planar substrate; and / or The flow chamber also includes one or more additional planar layers positioned above the recess in the planar substrate to form a closed channel; optionally, the one or more planar layers are attached to the planar substrate by pressing, adhesive bonding (preferably with a biocompatible adhesive, more preferably with silicone or silicone-based adhesives), solvent bonding, ultrasonic welding, thermal bonding, welding, or 3D printing.

40. The method of claim 36, wherein the planar substrate comprises a polymeric material, such as a cyclic olefin polymer or a cyclic olefin copolymer; and further comprises: A collection hole is formed on the planar substrate and is in fluid communication with the terminal portion of the outlet channel; and / or An internal channel inlet at a first hole height and an internal outlet at a second hole height, wherein the inlet is in fluid communication with the outlet channel of the flow chamber cartridge, and wherein the second hole height is higher than the first hole height.

41. The method of claim 37, wherein the planar substrate comprises a polymeric material, such as a cyclic olefin polymer or a cyclic olefin copolymer; and Also includes: A collection hole is formed on the planar substrate and is in fluid communication with the terminal portion of the outlet channel; and / or An internal channel inlet at a first hole height and an internal outlet at a second hole height, wherein the inlet is in fluid communication with the outlet channel of the flow chamber cartridge, and wherein the second hole height is higher than the first hole height.

42. A method for separating said live cells and / or cell nuclei from a mixture comprising live cells and / or cell nuclei, dead cells and nuclear fragments, said method comprising: A flow chamber cartridge is provided, the flow chamber cartridge including a processing channel and a plurality of outlet channels, wherein the outlet channels of the flow chamber cartridge have a volume larger than that of the processing channel; The sample solution containing live and dead cells, as well as paramagnetic compounds, flows into the processing channel; The flow chamber cartridge is placed in a magnetic field that is substantially parallel to and aligned with the processing channel; The processing channel and the sample contained therein are kept completely within the magnetic field in a stopped flow state for a sufficient time to allow live cells and dead cells to be separated at a vertical distance within the processing channel. as well as Simultaneously, sample fractions rich in live cells and / or cell nuclei and sample fractions rich in dead cells and cell nucleus debris are discharged into the outlet channel; The sample solution contains particles with a size of about 1 to about 15 micrometers; and wherein: a) The particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, in a manner that inhibits the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus; or b) The particles form complexes with cell debris, or form structures in the sample medium that interact with cell debris by increasing the movement of at least a portion of the cell debris in a selected direction relative to the movement of the cell nucleus.

43. The method of claim 42, wherein the particles form a complex with cell debris, or form a structure in the sample medium that interacts with cell debris, in a manner that inhibits the movement of at least a portion of the cell debris in the direction of environmental gravity relative to the cell nucleus.

44. The method of claim 43, further comprising providing a flow chamber cartridge that is substantially free of any liquid or paramagnetic compound prior to the introduction of the sample solution.

45. A kit for isolating cell nuclei, comprising: a) Cell nucleus separation buffer and one or more additional buffers, solvents, proteins, particles, enzymes and / or stabilizers.

46. ​​The kit of claim 45, wherein the separating particles comprise microbeads, such as polystyrene microbeads, optionally coated with a protein such as streptavidin, having a size of about 1 micrometer to about 10 micrometers; for example, a size of about 1 to about 8 micrometers; for example, a size of about 1 to about 5 micrometers; for example, a size of about 1, about 2, about 3, about 4 or about 5 micrometers.

47. The kit of claim 45 or claim 46, wherein the separated particles comprise microbeads with a size of about 1 to about 8 micrometers; for example, with a size of about 1 to about 5 micrometers; for example, with a size of about 1, about 2, about 3, about 4 or about 5 micrometers.

48. The kit according to claim 47, wherein the size of the separated particles is about 3 micrometers.

49. The kit according to any one of claims 45-48, wherein one or more components of the kit are provided in a solid form to facilitate reconfiguration.

50. The kit according to any one of claims 45-49, further comprising a gadolinium-containing suspending agent.

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