JP2009511001A - Device and method for magnetic concentration of cells and other particles - Google Patents

Abstract

The present invention relates to devices and methods for concentrating cells and other desired analytes by using a magnetic field alone or in combination with size separation. The devices and methods can be beneficially used to perform enrichment of rare cells, such as fetal cells or epithelial cells present in a sample, eg, maternal blood.
[Selection figure] None

Description

The present invention relates to the fields of cell separation, medical diagnosis, and microfluidic devices.

  It can often happen that clinically or environmentally valuable information is present in the sample but is too small to be detected. Accordingly, various enrichment or amplification methods are often used to increase the detectability of such information.

  In the case of cells, various flow cytometry and cell sorting methods are available, but these techniques generally use large and expensive devices, requiring large samples and skilled operators. Such cytometers and sorters use methods such as electrostatic deflection, centrifugation, fluorescence activated cell sorting (FACS), and magnetically activated cell sorting (MACS). To achieve cell separation. These methods often have the problem that the sample cannot be concentrated enough to allow analysis of rare components in the sample. Furthermore, such techniques may result in inconvenient loss of rare components, for example, due to inefficient separation or component decomposition.

  Accordingly, there is a need for new devices and methods for sample concentration.

SUMMARY OF THE INVENTION As a whole, the present invention takes advantage of magnetic properties and generally combines with another separation element, such as size, shape, deformability, and affinity, for cells and other analytes of interest. The present invention relates to a device and a method that enable concentration of sucrose. Preferably, the analyte of interest is separated based on its intrinsic magnetic properties, which may be varied as described herein.

  Accordingly, the present invention relates to a device that produces a sample in which a first cell or component thereof is concentrated relative to a second component, and a channel through which the first cell or component flows, as well as from 0.05 Tesla to 5. It relates to a device comprising a magnet that generates a magnetic field in the channel in the range of 0 Tesla and a magnetic gradient in the range of 100 Tesla / m to 1,000,000 Tesla / m. The first cell or component may be retained in the channel and the second component may not be retained in the channel, or vice versa. The channel may include first and second outlets, where the first cell or component thereof is directed toward the first outlet, while the second component is directed to the second outlet. The The device may further include a pump capable of generating a flow rate that causes more than 50,000 cells per second or components thereof to flow into the channel.

  The device may further include an analysis module that concentrates the first cell or component based on size, shape, deformability, or affinity. The analysis module includes, for example, a first channel having a structure that deterministically deflects particles having a hydraulic size larger than the critical size in a direction that is not parallel to the average flow direction in the structure. The structure may include an array of barrier structures that form a network of gaps. The fluid flowing through the gap is non-uniformly divided into a main flow and a sub-flow so that the average flow direction of the main flow is not parallel to the average fluid flow direction in the channel. The array of barrier structures may have first and second rows so that fluid flowing through one gap in the first row is unequally divided into two gaps in the second row. The second row is arranged transversely to the first row.

  The device may further comprise a reagent capable of changing the magnetic properties of the first cell or component or the second component. A reagent is a protein present, for example, in a first cell or component or in a second component that alters magnetic properties such as, for example, iron-containing fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome. . Typical examples of reagents include sodium nitrite, carbon dioxide, oxygen, carbon monoxide, and nitrogen. The reagent may further cause expression or overexpression of the magnetic protein in the first cell or component, or in the second component. For example, the reagent can transfect a first cell or second component with a magnetically responsive protein. The reagent may further comprise magnetic particles that bind to or are incorporated into the first cell or component or the second component.

  The first cell is, for example, a blood cell (for example, an adult nucleated red blood cell, or a fetal nucleated red blood cell from a fetus etc. less than 10 weeks), another nucleated cell, or a denucleated cell. The first cell can be of mammals, birds, reptiles, or amphibians. Typical examples of components of the first cell include the nucleus, perinuclear compartment, nuclear membrane, mitochondria, chloroplast, or cell membrane, lipid, polysaccharide, protein, nucleic acid, virus particle, and ribosome Is mentioned.

  In preferred embodiments, at least 90% of the first cells or components are retained in the device and at least 90% of the second component is not retained in the device.

  In another aspect, the device of the present invention is used to create a sample enriched with a first cell or component thereof relative to a second component. The method includes introducing a sample comprising a first cell or component into the channel and changing the path of the first cell or component or the second component relative to the other based on magnetic properties. Make a sample enriched with cells or components. The sample introduced into the device may concentrate the first cell or component relative to the third component. For example, the sample may be contacted with an analysis module that concentrates the first cell or component relative to the third component based on size, shape, deformability, ease of lysis, label, or affinity. A typical example of an analysis module is a first channel having a structure that deterministically deflects particles having a hydraulic size larger than the critical size in a direction that is not parallel to the average flow direction in the structure, Here, the particle is the first cell or component or the third component in the sample. The sample enriched in the first cells or components may retain at least 70% of the first cells or components present in the sample. The sample in which the first cells or components are concentrated is concentrated 100 times, for example. The method may further include contacting the sample with a reagent that can change the magnetic properties of the first cell or component or the second component. The sample enriched with the first cells or components includes at least 90% of the first cells or components in the introduced pre-concentration sample, and less than 10% of the second component in the sample before enrichment. May be included. Typical examples of reagents, first cells, their components, second components, purity, and flow rates are described herein.

  The invention further relates to an alternative method of making a sample enriched for a first cell or component thereof relative to a second component, wherein the method comprises the first cell or component as a potential. A denatured sample by contacting said sample with a reagent that alters the magnetic properties of the protein expressed in the first cell or component or second component in the sample as described herein, Contacting the denatured sample with a channel having a magnet that generates a magnetic field and magnetic gradient that is opposite the channel and that can change the path of the first cell or component or the second component relative to the other. To produce a sample enriched in the first cells or components. In some embodiments, the sample is enriched in the first cells or components relative to the third component prior to contacting the magnetic field. For example, the sample may concentrate the first cell or component relative to the third component. For example, an analysis module that concentrates the first cell or component relative to the third component based on size, shape, deformability, solubility, label, or affinity can be contacted. A typical example of an analysis module is a first channel having a structure that deterministically deflects particles having a hydraulic size larger than the critical size in a direction that is not parallel to the average flow direction in the structure, In this case, the particle is the first cell or component or the third component in the sample. Typical examples of structures are described herein. A sample enriched for the first cells or components will retain at least 70% of the first cells or components present in the sample. The sample in which the first cells or components are concentrated is concentrated, for example, 100 times or 1000 times. The sample enriched in the first cell or component has at least 90% of the first cell or component in the introduced pre-concentration sample, and less than 10% of the second component in the sample before enrichment. May be included. Typical examples of reagents, first cells, their components, second components, purity, and flow rates are described herein.

In another aspect, the present invention relates to a method for concentrating a first analyte of a fluid sample (eg, a blood sample such as maternal blood) relative to a second and third analyte, wherein the method comprises a hydraulic Based on the size, a first concentration that concentrates the first analyte from the fluid sample using a plurality of barriers that direct the first analyte in the first direction and the second analyte in the second direction. And performing a second concentration step that concentrates the first analyte from the fluid sample based on the intrinsic or external magnetic properties of the first or third analyte. A typical example of the first analyte is a cell as described herein. The second concentration step may include applying a magnetic field to the product obtained in the first concentration step. The magnetic field may attract or repel the first or third analyte. In general, the magnetic field changes the path of the first analyte relative to the third analyte. The method may further comprise changing the magnetic properties of the product of the first concentration step, for example by deoxygenation. The deoxygenation step may comprise contacting the product of the first concentration step with CO, CO ₂ , N ₂ , or NaNO ₂ . The method may further comprise paramagnetization or diamagnetization of the first or third analyte. The first concentration step and the second concentration step may be performed continuously. In some embodiments, the first concentration step or the second concentration step comprises a plurality of concentration steps that are performed sequentially or in parallel with each other. The first concentration step or the second concentration step is performed during sample flow. The second enrichment step may be based on intrinsic or external magnetic properties. Preferably, more than 50,000 analytes are subjected to concentration per second. Typical magnetic fields and magnetic gradients are described herein.

  The present invention further provides a hydrodynamic size smaller than the critical size in a first direction toward the first outlet with one or more first analytes having a hydrodynamic size greater than the critical size. A first module having an array of barrier structures that selectively guide one or more second analytes in a second direction toward the second outlet, the first module discharged from the first outlet; A system comprising a second module having a channel for receiving one analyte and a magnet for generating a magnetic field and a magnetic gradient for changing the course of the first analyte in the channel.

  The present invention further provides a hydrodynamic size smaller than the critical size in a first direction toward the first outlet with one or more first analytes having a hydrodynamic size greater than the critical size. A flow channel having an array of two-dimensionally arranged barrier structures that selectively guides one or more second analytes in a second direction toward the second outlet; The present invention relates to a system comprising a magnet for generating a magnetic field and a magnetic gradient for changing the course of an analyte.

  For example, the first analyte is nucleated red blood cells, such as fetal nucleated red blood cells, and the second analyte is, for example, enucleated red blood cells. As described herein, adult nucleated red blood cells can be used for the diagnosis and treatment of various diseases. Examples of the first analyte include fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome. The system may further include a reservoir connected to the array or channel of barrier structures and comprising an oxygen scavenger or other reagent capable of changing the magnetic properties. The system may further comprise a reservoir containing a probe, such as a nucleic acid probe or antibody probe, for specifically binding the first analyte or component thereof. The path of the first analyte varies based on, for example, intrinsic or external magnetic properties. Typical magnetic field strengths used in this system are between 0.5 Tesla and 5.0 Tesla and typical magnetic gradients are between 100 Tesla / m and 1,000,000 Tesla / m. . The system may further include a pump capable of generating a flow rate that causes more than 50,000 cells per second or components thereof to flow into the channel.

  In another aspect, the present invention provides a device for producing an analyte-enriched sample, which device has particles with a hydraulic size larger than the critical size relative to the average flow direction in the structure. A first channel (eg, a microfluidic channel) having a structure that deterministically deflects in a non-parallel direction, and a reservoir fluidly connected to the effluent of the first channel through which the analyte flows into the reservoir Wherein the particles are analyte particles or non-analyte components in the sample, and the reservoir may contain reagents that alter the magnetic properties of the analyte. The structure comprises, for example, an array of barrier structures that form a network of gaps, such that the fluid flowing through the gaps flows over the critical size in the main stream and particles smaller than the critical size in the side stream. The main stream and the secondary stream are divided unevenly. The array of barrier structures may have first and second rows, such that fluid flowing through one gap in the first row is diverted unevenly to two gaps in the second row. In addition, the second row is at a position shifted laterally with respect to the first row. The desired analyte may have a hydrodynamic size that exceeds or is less than the critical size. The device may include a magnet capable of generating a magnetic field, and further includes a magnetic barrier structure (eg, a barrier structure comprising a permanent magnet or a non-permanent magnet) disposed in a second channel (eg, a microfluidic channel). A region of a barrier structure comprising). The magnetic barrier structures may be arranged in a two-dimensional array. The reservoir of the device may further include a second channel having a magnet. A reagent (eg, sodium nitrite) may alter the intrinsic magnetic properties of one or more analytes. In one embodiment, a reagent, such as holotransferrin or magnetic particles, may bind to one or more analytes. The magnetic particles are further antibodies (eg anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, anti-fetal hemoglobin, anti-EpCAM, anti-E-cadherin, or anti-Muc-1) or their antigen binding properties. It may contain fragments.

  The present invention further provides a method of making a sample enriched with a first analyte relative to a second analyte, wherein the method includes particles having a hydrodynamic size larger than the critical size in the structure. Applying at least a portion of the sample to a device having a structure that deterministically deviates in a direction that is not parallel to the average flow direction, the first analyte is concentrated and the second analyte comprises a second analyte A first analyte that is denatured by combining a second sample with a reagent that alters the magnetic properties of the first analyte, and a magnetic field on the second sample To produce a sample enriched in the first analyte by generating a differential force that physically separates the first analyte, whose magnetic field has been denatured, from the second analyte. The Comprising a thing. The reagent may bind to the first analyte. In another embodiment, a reagent (eg, sodium nitrite) may change the intrinsic magnetic properties of the first analyte. In yet another aspect, the reagent may comprise magnetic particles that bind to or are incorporated into the first analyte. The magnetic particles may comprise an antibody (eg, anti-CD71, anti-GPA, anti-antigen i, anti-CD45, anti-CD34, anti-fetal hemoglobin, anti-EpCAM, anti-E-cadherin, or anti-Muc-1) or an antigen-binding fragment thereof. Good. The analyte may have a hydrodynamic size that exceeds or is less than the critical size. The sample may be a maternal blood sample. The first analyte may be a cell (eg, a bacterial cell, a fetal cell, or a blood cell such as a fetal red blood cell), an organelle (eg, a nucleus), or a virus.

  The present invention further provides a method of making a sample enriched in red blood cells for a second blood component (eg, maternal blood cells), wherein the method comprises a sample comprising red blood cells (eg, fetal red blood cells). Generating oxygenated hemoglobin upon contact with a reagent that oxidizes iron comprises applying a magnetic field to the sample, wherein red blood cells having oxidized hemoglobin are attracted to the magnetic field more strongly than the second blood component. Thus, a sample enriched with red blood cells is prepared. The method further includes, prior to the contacting step, at least a sample in a device having a structure that deterministically deflects particles having a hydraulic size larger than the critical size, for example, in a direction that is not parallel to the average flow direction in the structure. A sample may be enriched for red blood cells by applying a portion of (eg, concentrating fetal blood cells to maternal red blood cells).

  In another aspect, the invention provides a device for producing a sample enriched in red blood cells, the device comprising an analytical device that concentrates red blood cells based on size, shape, deformability, or affinity; and A reservoir is provided with a reagent that oxidizes iron, where the reagent (eg, sodium nitrite) increases the magnetic response of red blood cells. The analytical device may include a first channel having a structure that deterministically deflects particles having a hydraulic size larger than the critical size in a direction that is not parallel to the average flow direction in the structure.

  The invention further includes one or more binding sites that selectively bind to GPA, fetal hemoglobin, antigen i, antigen I, CD34, CD45, CD15, CD71, EGFR, or EpCAm (eg, antibodies such as monoclonal antibodies) And a reagent comprising a plurality of magnetic particles coupled to.

  These reagents can be used in a method of separating one or more cells of interest from a mixture of cells, combining the cell mixture with the reagent, and combining the cell mixture with the reagent. Incubating for a time sufficient for the site to selectively bind to one or more cells of interest in the mixture, as well as magnetic particles from cells that did not bind to the magnetic particles by applying a magnetic field to the mixture Separating the bound particles. The method may further comprise the step of enriching the mixture of cells for one or more cells of interest. This enrichment step may include size separation by an array of barrier structures or selective lysis of one or more cells that are not of interest.

  In another aspect, the present invention introduces a sample containing red blood cells into the column, applies a magnetic field to the column, and sets the path of the first type of red blood cells relative to the second type of red blood cells to a magnetic property. The present invention relates to a method for concentrating a subpopulation of red blood cells by making a change based on the first type, in which case the first type of red blood cells is more attracted to the magnetic field than the second type of red blood cells. The first type of red blood cell is, for example, a mature red blood cell or a positively dyed erythroblast, and the second type of red blood cell is, for example, a positively colored right erythroblast or a polychromatic right erythroblast. .

  The present invention further introduces a sample containing cells into the column, applies a magnetic field to the column, and determines the path of the cells containing the magnetically sensitive particles in the sample based on the magnetic properties. The present invention relates to a method for concentrating a population of cells resident in magnetically sensitive particles by varying the type of cells. Particles that are susceptible to magnetism are, for example, red blood cells or magnetotactic bacteria. The magnetically sensitive particles may include magnetite or greigite. The cells containing particles susceptible to magnetic influence are, for example, monocytes or macrophages. This method can be used to diagnose or monitor the treatment of familial hemophagocytic histiocytosis, acute monocytic leukemia, and lymphoma.

  The present invention further introduces a sample comprising blood cells into the column and applies a magnetic field to the column, and prior to, during or after the introduction, with leukocytes (eg, through anti-CD45 antibody or anti-CD15 antibody). By contacting the sample with a magnetically sensitive reagent to which it binds and changing the path of red blood cells and white blood cells in the sample relative to a third type of cells (eg, stem cells) based on magnetic properties, blood The present invention relates to a method for removing red blood cells and white blood cells in a sample (eg, cord blood).

  “Analyte” means a molecule, other chemical species, such as ions, or particles. Typical analytes include cells, viruses, nucleic acids, proteins, carbohydrates, and small organic molecules.

  By “biological particle” is meant any species that is derived from an organism and that does not dissolve in an aqueous medium on the time scale of sample collection, preparation, storage, and analysis. Examples include cells, particulate cellular components, viruses, and complexes comprising proteins, lipids, nucleic acids, and carbohydrates.

  By “biological sample” is meant any sample that contains or may contain biological samples or biological particles. A preferred biological sample is a cell sample.

  “Blood component” means any component of whole blood comprising host red blood cells, white blood cells, and platelets. Blood components further include plasma components such as proteins, lipids, nucleic acids, and carbohydrates, and other that may be present in the blood for reasons of current or past pregnancy, organ transplantation, disease, or infection, for example. Any cell is included.

  “Cell sample” means a sample comprising cells or components thereof. Such samples include natural fluids (eg blood, lymph, cerebrospinal fluid, urine, cervical lavage, water samples), some of such fluids, and cells introduced Liquid (culture solution, liquefied tissue sample) can be mentioned. The term also includes lysates.

  “Capture site” means a chemical species to which an analyte binds. The capture site may be a compound bound to the surface or a substance forming the surface. Typical capture sites include antibodies, oligopeptides or polypeptides, nucleic acids, other proteins, synthetic polymers, and carbohydrates.

  “Channel” means a gap through which fluid can flow. The channel may be a capillary, a conduit, or a strip of hydrophilic pattern on the lipophilic surface that encloses the aqueous liquid.

  By “component” of a cell is meant any component of the cell that can be at least partially isolated by lysis of the cell. Cell components include organelles (eg, nucleus, peri-nuclear compartment, nuclear membrane, mitochondria, chloroplast, or cell membrane), polymers or molecular complexes (eg, lipids, polysaccharides, membrane proteins). , Transmembrane proteins, cytoplasmic proteins, natural nucleic acids, therapeutic nucleic acids, pathogenic nucleic acids, viral particles, or ribosomes), or other molecules (eg, hormones, ions, cofactors, or drugs). By “component” of a cell sample is meant a subset of cells contained in that sample.

  "Concentrated sample" means a sample that contains an analyte that has been processed to increase the amount of that analyte relative to other analytes typically present in the sample. To do. For example, increase the amount of analyte of interest by at least 10%, 25%, 50%, 75%, or 100%, or at least 1000 times, 10,000 times, 100,000 times, or 1,000,000 times By increasing the value, the sample can be concentrated.

  By “removed sample” is meant a sample comprising an analyte that has been processed to reduce the amount of that analyte relative to other analytes typically present in the sample. For example, removing the sample reduces the amount of analyte of interest by at least 5%, 10%, 25%, 50%, 75%, 90%, 95%, 97%, 98%, 99%, or 100% You may let them.

  An “exchange buffer” in the context of a sample (eg, a cell sample) is a medium other than the medium in which the sample is originally suspended, into which one or more components in the sample are contained. Means the medium to be exchanged.

  By “external magnetic property” of an analyte is meant a magnetic property that is not intrinsic to the analyte.

  “Flow extraction boundary” means a boundary designed to remove fluid from the array.

  By “flow supply boundary” is meant a boundary designed to add fluid to the array.

  “Gap” means an opening through which fluid and / or particles can flow. For example, the gap may be a capillary, a space between two barrier structures through which fluid can flow, or a hydrophilic pattern on an oleophilic surface that traps aqueous fluid. In a preferred embodiment of the invention, the gap network is determined by an array of barrier structures. In this embodiment, the gap is a space between adjacent barrier structures. In a preferred embodiment, the interstitial network is constructed by an array of barrier structures on the substrate surface.

  “Hydraulic size” means the effective size of a particle when interacting with flow, barrier structure (eg, columnar), or other particles. This barrier structure or other particle may be a microfluidic structure. This term is used as a general term for the volume, shape, and deformability of a flowing particle.

  “Intracellular activation” means activation of a second messenger pathway that causes activation of a transcription factor, or activation of a kinase or other metabolic pathway. Intracellular activation due to changes in extracellular membrane antigens can also cause changes in receptor trafficking.

  An “inherent magnetic property” of an analyte means a magnetic property that is intrinsic to the analyte. Intrinsic magnetic properties may exist from the beginning of the analysis or may be induced in the analyte by a suitable reagent. Typical intrinsic magnetic properties include those introduced by iron-containing proteins expressed by cells.

  A “labeling reagent” can be bound, internalized, or absorbed by an analyte, eg, shape, form, color, fluorescence, luminescence, phosphorescence, absorbance, magnetic properties, or Means a reagent detectable by radiation.

  “Metabolome” refers to a set of compounds in a cell that are involved in metabolic reactions and exclude proteins and nucleic acids that are required for cell maintenance, growth, or normal function.

  “Microfluidic” means that at least one dimension is less than 1 mm.

  “Barrier structure” means an obstacle to flow in a channel, such as a protrusion from one surface. The barrier structure may be, for example, a column protruding from the substrate, or a hydrophobic barrier in the case of an aqueous fluid. In some embodiments, the barrier structure may be partially permeable. For example, the barrier structure may be a columnar portion made of a porous material. In this case, the pores allow the aqueous component to permeate, but the particles to be separated are too small to enter.

  “Shrinkage reagent” means a reagent that reduces the hydrodynamic size of particles. Contractile reagents can act by reducing the volume of the particles, increasing deformability, or changing shape.

  By “swelling reagent” is meant a reagent that increases the hydrodynamic size of the particles. Swelling reagents can act by increasing the volume of the particles, decreasing the deformability, or changing the shape.

  “Substantially larger” means at least 2 times, 3 times, 5 times, 10 times, 25 times, 50 times, or 100 times larger.

  “Substantially smaller” means at least 2 times, 3 times, 5 times, 10 times, 25 times, 50 times, or 100 times smaller.

  Other features and advantages will be apparent from the following description and the claims.

Detailed Description of the Invention The present invention provides analytical devices and methods useful for concentrating analytes in a sample. In general, concentration occurs through the interaction of the analyte or other components of the sample with the magnetic field. Analytes may be based on intrinsic magnetic properties (eg, iron-containing proteins), external magnetic properties (eg, magnetic beads bound to the analyte), or lack of intrinsic or external magnetic properties. It can be concentrated. Concentration can be based on the existing magnetic properties of the components in the sample, or can be based on reaction with a reagent that can change the magnetic properties (eg, induction or addition). The methods and devices of the present invention can be used to make concentrated samples of analytes such as red blood cells (eg, fetal red blood cells from maternal blood).

Remaining components in the sample using magnetic separation, eg, the intrinsic magnetic properties of the analyte as altered in the methods described herein, or the external magnetic properties of the analyte, eg, by providing magnetic beads The analyte can be isolated, concentrated, or removed from the. Such isolation, enrichment, or removal can be achieved by positive selection, i.e. selection by which the required analyte is attracted to a magnetic field, or negative selection, i.e. by repelling or not being affected, It may include sorting that is not attracted to the magnetic field. In either case, a population of analytes containing the desired analyte can be recovered for analysis or further processing.

  The device used to perform the magnetic separation can be any device that can generate a magnetic field. In one embodiment, the magnetically responsive analyte is concentrated using a MACS column (eg, Miltenyi Biotec). For example, if the analyte is magnetically responsive by reaction with the reagents described herein, the analyte is attracted to the MACS column in the presence of a magnetic field, thereby making the desired component with respect to the remaining components in the sample. It is possible to concentrate the analyte. In another aspect, concentration can be achieved using a device comprising a plurality of magnetic barrier structures, typically a microfluidic device. If the analyte in the sample is magnetically responsive (eg, through reaction with a reagent that changes the intrinsic magnetic properties of the analyte or by binding of magnetically responsive particles to the analyte), the analyte is a barrier. Can bind to the structure, thereby allowing concentration of the bound analyte. Alternatively, negative selection may be incorporated as another option. In this example, the desired analyte may be non-magnetic responsive or non-magnetic responsive, or the unwanted analyte may be magnetic responsive, magnetic responsive, or It may be combined with magnetically responsive particles. In this case, the sample is concentrated for the desired analyte because one or more unwanted analytes are retained in the magnetic device and not the desired analyte.

  In another embodiment, the sample is treated with a reagent comprising magnetic particles prior to applying the magnetic field. As described herein, the magnetic particles may be coated with a suitable capture site to which an analyte such as an antibody can bind. Application of a magnetic field to the treated sample selectively attracts analyte bound to the magnetic particles.

Other regions of the channel or device may or may not be magnetically responsive. In one embodiment, the channel through which the analyte passes is coupled to a magnet capable of generating an appropriate magnetic field within the channel. A typical magnet is shown in FIG. Or as another option, the channel in the device comprises a magnetically responsive region that typically changes the applied magnetic field. In general, the magnetic field strength is 5.0 Tesla, for example, about 0.5 Tesla, from 0.05 Tesla, the magnetic-responsive region 100 Tesla / m to 1,000,000 Tesla / m, for example about ¹⁰⁴ Tesla A magnetic gradient of / m is generated.

  The magnetic region of the device can be manufactured using hard or soft magnetic materials, examples of which include iron, steel, nickel, cobalt, rare earths, neodymium-iron-boron, iron-chromium- Examples include, but are not limited to, cobalt, nickel-iron, cobalt-platinum, and strontium ferrite. Part of the device may be manufactured directly from a magnetic material, or the magnetic material may be applied to another material. Hard magnetic materials can generate a magnetic field without any other manipulation, thus simplifying device design. On the other hand, a soft magnetic material can be released and processed downstream by simply demagnetizing the material. Depending on the type of magnetic material, the coating method may include cathodic sputtering, sintering, electrolytic deposition, or thin film coating of a polymer binder-magnetic powder composite. A preferred embodiment is a thin film of a microfabricated barrier structure (eg, silicon pillar) by a spin casting method of a polymer composite such as polyimide-strontium ferrite (polyimide serves as a binder and strontium ferrite serves as a magnetic filler). It is a coating. After coating, the polymer magnetic coating is cured to achieve stable mechanical properties. After curing, the device is briefly exposed to an externally induced magnetic field that determines the preferred direction of permanent magnetism of the device. The magnetic flux density and intrinsic coercivity of the magnetic field from the barrier structure can be controlled by the volume percentage of the magnetic filler.

  In another aspect, the conductive material is micropatterned on the outer surface of the sealed microfluidic device. The pattern may consist of a single electrical circuit having a spatial periodicity of about 100 microns. By controlling the layout of this electric circuit and the current intensity flowing through this circuit, it is possible to establish a periodic structure of a region having a high magnetic field strength and a region having a low magnetic field strength in a sealed microfluidic device.

  In yet another embodiment, the magnetically responsive region comprises filled iron beads coated with non-sticky plastic or Teflon.

  In any of the above-described embodiments, any magnetic field generation source may be used in the present invention, and examples of the generation source include a hard magnet, a soft magnet, an electromagnet, a superconducting magnet, or a combination thereof. In one embodiment, spatially non-uniform permanent magnets or electromagnets are used to form an ordered and possibly periodic array of magnetic particles in an originally unpatterned microfluidic channel. (Deng et al., Applied Physics Letters, Vol. 78, p. 1775, (2001)). Alternatively, a non-uniform magnetic field that does not have regular periodicity may be used. An electromagnet can also be used to generate a non-uniform magnetic field in the device. Regions with high and low magnetic field strength are created by the non-uniform magnetic field, and as a result, magnetic particles are attracted with a periodic arrangement. Other external magnetic fields can be used to form a magnetic region that attracts magnetic particles. Hard magnetic materials may be used in the manufacture of the device, thereby eliminating the need for an electromagnet or external magnetic field. In one embodiment, the device comprises a plurality of channels having magnetic regions, for example to increase processing capacity. Furthermore, these channels may be stacked vertically.

  In the embodiments described above, the analyte associated with the magnet can be released from a predetermined location in the channel, for example by increasing the flow velocity of the entire fluid flowing through the device, weakening the magnetic field, or some combination of the two.

For example, the magnetic field can be adjusted to affect supermagnetic and paramagnetic particles having a mass susceptibility in the range of 0.1-200 × 10 ⁻⁶ m ³ / kg. Useful paramagnetic particles can be classified according to their size into fine particle form (cell size of 1 μm to 5 μm), colloidal form (on the order of 100 nm), and molecular form (on the order of 2 nm to 10 nm). The basic force acting on the paramagnetic material is expressed as shown in equation (1).

Where F _b is the magnetic force acting on the paramagnetic material of volume V _b , Δχ is the difference between the magnetic susceptibility χb of the magnetic particles and the magnetic susceptibility χf of the medium surrounding it, μ ₀ is the permeability in free space, and B is the external magnetic field And ∇ represents the gradient operator. A wide range of particulate, colloidal, and molecular paramagnetic materials can be attracted and retained by controlling and adjusting the magnetic field.

Reagent capable of altering magnetic properties In some embodiments, an analyte or other sample component can alter the intrinsic and external magnetic properties of that analyte or other sample component. Reacts with reagents. The exact nature of the reagent depends on whether the nature of the analyte or the magnetic properties that the reagent changes are intrinsic or external. Typical reagents include reagents that oxidize or reduce transition metals, magnetic beads that can bind to analytes, or iron (eg, as described in US Pat. No. 4,508,625), other magnetic materials, or Examples include reagents that can be chelated or bound to magnetic particles. Specific reagents include chemicals such as sodium nitrite, gases such as nitrogen, oxygen, carbon dioxide, carbon monoxide, and mixtures thereof. For example, the reagent can act to make the analyte paramagnetic or diamagnetic. The reagent can further act to deoxygenate analytes such as myoglobin or hemoglobin. Reagents enable, reduce, or increase the attractiveness of an analyte to a magnetic field by changing the magnetic properties of the analyte, and enable, decrease, or increase the repulsion to a magnetic field. Or to remove the magnetic properties of the analyte so that it is not affected by the magnetic field. The reagent can further alter the magnetic properties of the fluid in which the analyte is lysed, suspended, or otherwise carried, or the cytosol of cells. The reagent can further change the rheology of the analyte.

  In some embodiments, the magnetic particles bind to the analyte to provide an external magnetic response. In these aspects, any particle that responds to a magnetic field can be used in the devices and methods of the present invention. Desirable particles are particles having a surface chemistry that can be chemically or physically modified, for example, by chemical reaction, physical adsorption, entanglement, or electrostatic interaction. The magnetic particles of the present invention may have any size and / or shape. In some embodiments, the magnetic particle size is less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, or less than 50 nm. In some embodiments, the magnetic particle size is 10 nm to 1000 nm, 20 nm to 800 nm, 30 nm to 600 nm, 40 nm to 400 nm, or 50 nm to 200 nm. In some embodiments, the magnetic particle size is greater than 10 nm, greater than 50 nm, greater than 100 nm, greater than 200 nm, greater than 500 nm, greater than 1000 nm, or greater than 5000 nm. The magnetic particles may be dry or liquid. Mixing the fluid sample with the second liquid medium containing magnetic particles may be done by any means known in the art.

  The capture site may be bound to the magnetic particle by any means known in the art. Examples include chemical reaction, physical adsorption, entanglement, or electrostatic interaction. The capture site that binds to the magnetic particle depends on the nature of the analyte of interest. Examples of capture sites include proteins (antibodies, avidin, cell surface receptors, etc.), charged or uncharged polymers (polypeptides, nucleic acids, synthetic polymers, etc.), hydrophobic or hydrophilic polymers, small molecules (biotin, receptors) Ligands, chelating agents, etc.) and ions, but are not limited to these. Using such capture sites, cells (eg, bacterial cells, pathogen cells, fetal cells, fetal blood cells, cancer cells, epithelial cells, endothelial cells, blood cells), organelles (eg, nuclei), viruses , Peptides, proteins, polymers, nucleic acids, supramolecular complexes, other biomolecules (eg organic or inorganic molecules), small molecules, ions, or combinations or fragments thereof. Specific examples of capture sites include anti-CD71, anti-CD36, anti-GPA, anti-EpCAM, anti-E cadherin, anti-Muc-1, and holotransferrin. Other specific antibodies are described herein. In another embodiment, the capture site is specific for fetal cells (eg, fetal red blood cells), cancer cells, or epithelial cells.

  The sample may further be combined with reagents that alter the magnetic properties inherent in the analyte. The denatured analyte may be modified to some extent magnetically responsive to the reagent as compared to the non-denatured analyte, or may be denatured to non-magnetic responsiveness. In one example, a sample containing fetal red blood cells (fRBC) (eg, a maternal blood sample from which maternal red blood cells have been removed) is treated with sodium nitrite, thereby oxidizing fetal hemoglobin in the fRBC. This oxidation changes the magnetic response of fetal hemoglobin to other components in the sample, such as maternal white blood cells, thereby allowing separation of fRBC. Further, fetal and maternal nucleated RBCs can be separated using differential oxidation of fetal and maternal cells. Any cell comprising a magnetically responsive component such as iron in hemoglobin (eg, adult or fetal), myoglobin, or cytochrome (eg, cytochrome C) is modified to produce the cell or its component (eg, small) The intrinsic magnetic response of an analyte such as an organ can be changed.

  In addition, the cells may be contacted with reagents that induce, block, promote or suppress the expression of magnetically responsive proteins or other molecules. For example, a vector encoding a magnetically sensitive protein or subunit (eg, hemoglobin or heme) is transfected with the primary gene of interest. Cells that have been successfully transfected can then be concentrated using the magnetic devices described herein. By such a method, transiently transfected objects can be quickly isolated before loss of function, or a stably transfected population can be ensured. It can also be manipulated so that the expression of proteins that are susceptible to magnetism depends on environmental conditions or the presence or absence of activators. Such reagents can be used to trigger expression in cells having native but inactive magnetically sensitive proteins.

Multi-Mode Magnetic System Concentration Using the method of the present invention, cells or other particles can be concentrated simultaneously based on intrinsic and external magnetic properties. For example, in a blood sample, red blood cells can be separated based on their intrinsic magnetic properties, while white blood cells can be separated after treatment with magnetic beads that bind to white blood cells through, for example, CD45 or CD15 antibodies. Such a multi-mode method allows the concentration of non-magnetically responsive cells or components of the sample. For example, cord blood stem cells can be separated from both red and white blood cells.

Analysis Device The device of the present invention can be used in connection with any analysis device or may include any analysis device. Examples include affinity columns, cell counters, particle sorters such as fluorescence activated and magnetic activated cell sorters, capillary electrophoresis devices, sample storage devices, and sample preparation devices. Microfluidic devices are particularly important in the context of the systems described herein.

  Typical analytical devices include, for example, International Publication No. 2004/029221, International Publication No. 2004/113877, Huang et al., Science, Vol. 304, p. 987-990 (2004), US Patent Publication No. 2004/0144651, US Patent Publication No. 5,837,115, US Patent Publication No. 6,692,952, US Patent Application No. 11 / 449,161, Size, shape, or deformability comprising filters, sieves, and deterministic separation devices as described in US Patent Application No. 11 / 227,904 and US Patent Application No. 11 / 449,149 Devices useful for separating particles based on their affinity; devices useful for affinity-based capture as described, for example, in WO 2004/029221 and US 2005/0266433; No. 2004/029221, U.S. Patent Publication No. 5,641,628, and U.S. Patent Application No. 11 / 449,149. Devices useful for the selective lysis of cells in a sample as described; and, for example, WO 2004/029221, U.S. Patent 6,692,952, U.S. Patent Publication 2004/0166555, and U.S. Patents. Devices useful for cell alignment as described in published application 2006/0128006 are included.

  In certain embodiments, an analytical device can be used to concentrate various analytes in a sample, eg, for recovery or further analysis. Rare cells or components thereof may be retained in the device or may be enriched relative to other cells as described, for example, in WO 2004/029221. Typical rare cells, depending on the sample, are fetal cells (eg, fetal red blood cells); stem cells (eg, undifferentiated stem cells); cancer cells; immune system cells (host or graft); epithelial cells; connective tissue Cells; bacteria; fungi; viruses; and pathogens (eg, bacteria or protozoa). Such rare cells can be isolated from samples comprising body fluids such as blood, or environmental source samples such as water or air samples. Fetal erythrocytes can be enriched from maternal peripheral blood, for example, to determine the sex of the developing fetus and to identify genetic features such as aneuploids or mutations. Cancer cells can also be enriched from peripheral blood for diagnosis and monitoring of treatment progress. Body fluids or environmental samples can also be screened for pathogens such as, for example, coliforms, blood-derived diseases such as sepsis, or bacterial or viral meningitis. Rare cells also include cells of one organism that are present in another organism, such as cells of a transplanted organ. For example, analytes retained or concentrated in the device can be labeled with, for example, fluorescent or radiological probes, subjected to chemical or genetic analysis (such as fluorescent in situ hybridization), and biological If it is, it can culture | cultivate, or can also follow by observation or a probe.

  The analytical device may or may not include a microfluidic channel, i.e. it may or may not be a microfluidic device. The dimensions of the channel of the device into which the analyte is introduced will depend on the size or type of analyte being handled. Preferably, the channel of the analytical device has at least one dimension (e.g. height, width, length or radius) of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5. , 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 mm or less. The microfluidic system used in the devices and methods described herein preferably has at least one dimension of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, Less than 0.3, 0.2, 0.1, or 0.05 mm. The preferred dimensions of the analytical device can be determined by one skilled in the art based on the desired application.

  Analytical devices (eg, deterministic devices) include reagents that can change the magnetic properties of the analyte (eg, cells such as red blood cells) (eg, magnetic particles or sodium nitrite with binding sites) A reservoir may be connected, or the reservoir may be included. The reservoir may include a channel, such as a microfluidic channel, a tube, or any other container that can receive an analyte and contact it with a reagent. The reservoir may be separable from the analysis device and may be integrated with the analysis device. Mixing of the reagent with the analyte may be done by any means including diffusion, mechanical mixing, chaotic mixing, convection, or turbulence. The reagent may be stored in a dry state in the reservoir and liquefied upon introduction of the sample, or stored as a solution and mixed with the sample. In another embodiment, the reagent is added to the reservoir by continuous or discontinuous input as the sample is introduced.

  The reservoir may further include a structure that allows separation of the denatured analyte from unreacted reagents or byproducts of the reaction of the reagent with the analyte. For example, deterministic separation can be utilized for this purpose, as described herein. Alternatively, a filter, rinse, or other means can be utilized as another option. Such a structure may or may not be included as part of the reservoir or analytical device.

  In one embodiment, the reservoir comprises a channel having a magnetic region on the surface that is patterned so that analytes flowing through the channel can contact, for example, by adhering magnetic particles to regions within the channel. For example, by appropriately selecting parameters such as magnetic particle size and shape relative to the channel dimensions, a pattern that facilitates the interaction between the analyte and the bound magnetic particles can be provided. Magnetic particles have an affinity such as an antibody (eg, anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, anti-fetal hemoglobin, anti-EpCAM, anti-E-cadherin, or anti-Muc-1). It may be covered by a suitable capture site that can bind to the analyte by a based mechanism. The magnetic particles may be uniformly disposed within the device or may be disposed within spatially separated regions. Furthermore, the structure may be fabricated in the device using magnetic particles. For example, by using two magnetic regions facing each other in the channel, it is possible to form a “bridge structure” that attracts magnetic particles and connects the two regions. The magnetic particles may adhere to the hard magnetic region of the channel or may adhere to a soft magnetic region that is activated to generate a magnetic field.

  An example of a reservoir is shown in FIG. 74, showing the geometry of the reservoir and the functional step flow of isolating the analyte of interest, eg, a cell or molecule, from a complex mixture and then releasing it. As shown, the reservoir has a barrier structure that extends from one surface of the channel toward the opposite surface. This barrier structure may or may not extend over the entire length of the channel cross section. In this example, the barrier structure is magnetic (eg, comprising a hard or soft magnetic material, or a position in a strong magnetic field in a non-uniform magnetic field) and attracts and holds magnetic particles, in which case The magnetic particles may be covered by capture sites or cells that are attracted to a magnetic field. Reservoir geometry, barrier structure distribution, shape, size, and flow parameters, for example, to optimize the efficiency of concentration of analytes of interest by attracting analytes bound to magnetic particles with capture sites (E.g., described in International Publication No. 2004/029221). One specific example is a wafer capped by an anodic reaction with a magnetic barrier structure having microstructures of various shapes (columnar, rectangular, trapezoidal or polymorphic) and sizes (10 to 999 microns). Are placed in a unique array (arrangement spacing and density vary between equilateral triangle array, diagonal array, and random array distribution), and denatured or native analytes within the continuously perfused fluid flow The collision frequency with the barrier structure is maximized. The exact geometry of the magnetic barrier structure and the distribution of the barrier structure will depend on the type of analyte being isolated, concentrated, or purified.

  FIG. 75 shows an example of the manufacture and functionalization of the reservoir. The magnetized barrier structure allows the reservoir to be adjusted after sealing. Due to the incompatibility of semiconductor processing parameters (high temperature heating or solvent-based sealants for bonding lids) and capture sites (sensitive to temperature and inorganic and organic solvents), such devices are versatile. Suitable for functionalization of any capture site. The ability to retain the capture site on a barrier structure (eg, a column) by using a magnetic field is an added advantage over the prior art that uses complex surface chemistry for immobilization. Such a reservoir allows the end user to easily and quickly fill the reservoir with the optimal capture site or mixture of capture sites, thereby expanding the variety of applications. This reservoir allows for on-demand and “just-in-time” one-step functionalization, which would result if the capture site was accompanied by chemical cross-linking at the manufacturing stage. The problem of site storage stability can be avoided. Capture sites that can be loaded and retained on the barrier structure include differentiation cluster (CD) receptors on all mammalian cells, cell receptor synthesis and recombinant ligands, and others that can be added to any magnetic particle But are not limited to organic molecules, inorganic molecules or compounds of interest.

Additional components The devices of the present invention may further comprise elements for example for the isolation, recovery, manipulation or detection of the analyte. Such elements are known in the art. For example, the device of the present invention (eg, a device incorporating a deterministic device) may further include other types of separation components comprising affinity separation, lysis separation, electrophoretic separation, centrifugation, and dielectrophoretic separation. May be included. The device of the present invention may further comprise components for two-dimensional imaging of the output from the device, such as an array or plane of wells. Such an array or plane of wells can be imaged or viewed by a microscope or other imaging device, such as a camera.

  The device of the present invention may further be used with other concentration devices on the same device or on another device. As for other concentration techniques, for example, International Publication No. 2004/029221, International Publication No. 2004/113877, US Patent Publication No. 6,692,952, US Patent Publication No. 2005/0282293, US Patent Publication. No. 2005/0266433 and US patent application Ser. No. 11 / 449,161, both of which are incorporated by reference.

Deterministic separation
In one embodiment, the present invention comprises channels and magnets that deterministically guide particles based on hydraulic size, for example, in combination with a reservoir comprising a reagent that can alter the magnetic properties of the particles. Provide the device. The present invention further creates a second sample enriched in the first analyte by applying the sample to a device comprising a channel that deterministically guides particles based on hydrodynamic size, Combine the second sample with a reagent that alters the magnetic properties of the first analyte or utilize existing magnetic properties and apply a magnetic field to separate the first analyte from the second analyte This provides a method for making a sample enriched for the first analyte relative to the second analyte.

  As one example, the channel comprises one or more arrays of barrier structures that move fluid components deterministically laterally. Such devices are described in Huang et al., Science, Vol. 304, p. 987-990 (2004), U.S. Patent Publication No. 2004/0144651, and International Publication No. 2004/029221. These devices may also use an array of gap networks, in which case fluid flowing through one gap is diverted unevenly to subsequent gaps. In one embodiment, the fluid flowing through the gap is diverted unevenly even though the gap is the same size. The fluid flow transports the particles to be separated through the array of gaps. This flow is arranged with a small angle (flow angle) with respect to the line of sight of the array. Particles with a hydrodynamic size larger than the critical size move along the line of sight of the array, while particles with a hydrodynamic size less than the critical size follow a flow in another direction. The fluid in the device flows under laminar conditions.

  The critical size is a function of several design parameters. Referring to the array of barrier structures shown in FIG. 1, each column of the barrier structures is shifted laterally by Δλ with respect to the previous column, where λ is the center-to-center distance between the barrier structures (FIG. 1A). ). The parameter Δλ / λ (“branch ratio”, ε) determines the ratio of the flow branched to the left side of the next barrier structure. In FIG. 1, ε is 1/3 for convenience of illustration. In general, if the flux flowing through the gap between the two barrier structures is φ, the secondary flux is εφ and the main flux is (1−εφ) (FIG. 2). In this example, the flux flowing through the gap is substantially diverted by one third (FIG. 1B). Each of the three fluxes flowing through the gap flows so as to sew around the barrier structure, but the average flow direction of each flux is the direction of the entire flow. FIG. 1C shows critical sizes (eg 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, or 30 microns). Such analytes are successively transferred to the main flux flowing through each gap and move together with the main flux.

Referring to FIG. 2, the critical size is about 2R _critical , where R _critical represents the distance between the stagnant flow line and the barrier structure. For example, if the center of mass of a particle such as a cell is outside the R _critical , the particle will move along the line of sight of the array according to the main flux. If the flow profile in the longitudinal section of the gap is known, R _critical can be determined (FIG. 3). It is the thickness of the fluid layer that makes up the side flux. For any gap size d, R _critical can be adjusted based on the branching ratio ε. Generally, when ε is small, R _critical is also small.

  In an array for deterministic lateral movement, differently shaped particles behave as if they are different in size (FIG. 4). For example, lymphocytes have a spherical shape with a diameter of 5 μm or less, and red blood cells have a biconcave disk shape with a diameter of 7 μm or less and a thickness of 1.5 μm or less. The long axis (diameter) of erythrocytes is longer than that of lymphocytes, but the short axis (thickness) is shorter than that of lymphocytes. When erythrocytes flow through an array of barrier structures by flow, if their long axes are aligned with flow, the hydrodynamic size is substantially the thickness (1.5 μm or less), which is smaller than the lymphocytes. When erythrocytes pass between arrays of barrier structures by hydrodynamic flow, their long axes tend to align with the flow and behave like particles approximately 1.5 μm wide that are “smaller” than lymphocytes. . Thus, the method and device may be the same in volume, but can separate analytes according to their shape. Furthermore, analytes with different deformability also behave as if they are different in size (FIG. 5). For example, two unmodified analytes of the same shape can be separated by deterministic lateral movement because one can deform more easily than the other when deformed in contact with the barrier structure of the array. Can do. Thus, separation within the device can be achieved based on any parameter that affects the physical dimensions, shape, and hydrodynamic size of the deformability including the deformability.

Referring to FIG. 6, as schematically represented, a critical size 2R can be obtained by feeding a mixture of analytes of different hydrodynamic sizes, such as cells, from the top of the array and collecting the analyte at the bottom. effluent and effluent containing less cells than critical size containing an analyte of magnitude greater than _critical, it is possible to obtain two kinds of effluent. Only one effluent may be collected, or both may be collected, for example when fractionating a sample into two or more subsamples. Analytes larger than the gap size will be captured in the array. Thus, the array has an operable size range. To be distributed to the main flux, the cells must be larger than the cut-off size (2R _critical ) and less than the maximum passage size (array gap size). The “size range” of the array is defined as the ratio of the maximum pass size to the cutoff size.

Separation of free unreacted reagent from denatured analyte A deterministic device can be used to separate free unreacted reagent from denatured analyte. As shown in FIG. 60, a labeling agent such as an antibody may be preincubated with an analyte (eg, a cell sample) prior to introduction into the deterministic device or within the deterministic device. The reagent desirably reacts specifically with the analyte of interest, eg, a cell population such as epithelial cells. Typical labeling agents include antibodies, quantum dots, phages, aptamers, fluorophore-containing molecules, enzymes capable of causing a detectable chemical reaction, reagents that change magnetic properties (eg, sodium nitrite), Or a functionalized bead is mentioned. In general, the reagent is smaller than the target analyte (eg, cell) or target analyte bound to the beads; thus, when a sample combined with the reagent is introduced into the device, the unreacted reagent is diverted Without passing through the device, the denatured analyte (eg, the analyte bound to the reagent) is deflected, thereby separating unreacted reagent from the denatured analyte. Conveniently, the method achieves both size separation and separation of free unreacted reagent from the analyte. In addition, the separation method facilitates downstream sample analysis, if desired, without the use of a release step or potentially disruptive analysis method, as described below.

  FIG. 61 shows the special case where a concentrated and labeled sample contains a non-target cell population that is separated along with the target cells to approximate size. This reagent selectively binds to the target cells so that non-target cells do not interfere with downstream sample analysis that relies on detection of bound labeling agent.

Array design deterministic separation can be achieved by using an array of gaps and barrier structures in the channel. Typical configurations for such arrays, bypass channels, and interfaces are described below.

Single-stage array In one embodiment, the single-stage stage comprises an array of, for example, cylindrical columnar barrier structures (FIG. 1D). In some embodiments, for example when separating white blood cells from red blood cells, the array has a maximum passage size that is several times greater than the cutoff size. This result can be achieved by a combination of a large gap size d and a small branching ratio ε. In a preferred embodiment, ε is 1/2 or less, for example, 1/3 or less, 1/10 or less, 1/30 or less, 1/100 or less, 1/300 or less, or 1/1000 or less. In such embodiments, the shape of the barrier structure can affect the flow profile in the gap; while the barrier structure may be shortened in the flow direction to shorten the array (FIG. 1E). The single stage array may include a bypass channel as described herein.

In another embodiment of a multistage array , multiple stages are used to separate analytes over a wide size range. A typical device is shown in FIG. The device shown in the figure has three stages, but any number of stages can be used, and the array may have as many stages as necessary. In general, the cutoff size of the first stage is larger than the cutoff size of the second stage, and the cutoff size of the first stage is smaller than the maximum passing size of the second stage (FIG. 8). The same applies to subsequent stages. In the first stage, for example, an analyte that may cause clogging in the second stage is deflected (and removed) before reaching the second stage. Similarly, the second stage deflects (and removes) analytes that may cause clogging in the third stage before reaching the third stage.

  As already mentioned, in a multi-stage array, large particle size analytes, such as cells, that can be clogged downstream are first deflected, and such deflected analytes avoid clogging. Therefore, it is necessary to bypass the downstream stage. Thus, the device of the present invention may include a bypass channel that removes products from the array. Although the bypass channel is described herein in terms of removing analytes that exceed a critical size, the bypass channel can be used to remove product from any part of the array.

  The different bypass channel designs are shown below.

Single Bypass Channel In this design, all stages share one bypass channel or have only one stage. One physical boundary of the bypass channel may be an array boundary and the other a sidewall (FIGS. 9 to 11). A single bypass channel can also be used with a dual array (FIG. 12).

  A single bypass channel can also be designed in conjunction with an example to maintain constant flux across the device (FIG. 13). As shown in the figure, the flux is maintained constant throughout the entire stage by changing the width of the bypass channel so that the flow in the channel does not interfere with the flow in the array. Such a design can also be used with dual arrays (FIG. 14). A single bypass channel can also be designed in conjunction with the array to maintain a substantially constant fluid resistance throughout the entire stage (FIG. 15). Such a design can also be used with dual arrays (FIG. 16).

Dual Bypass Channel In this design (FIG. 17), each stage has its own bypass channel, and the channels are separated from each other by side walls. Large particle size analytes, such as cells, are deflected to the main flux and go to the lower right corner of the first stage and then enter the bypass channel (bypass channel 1 in FIG. 17). Small cells that do not clog in the second stage are sent to the second stage, and cells larger than the critical size of the second stage are deflected to the lower right corner of the second stage and enter another bypass channel (FIG. 17). Bypass channel 2). You can repeat this design for as many stages as you need. In this aspect, the bypass channels are not fluidly connected, and a plurality of fractions can be collected or treated. The bypass channels need not be straight and need not be physically parallel to each other (FIG. 18). Dual bypass channels can also be used with dual arrays (FIG. 19).

  The dual bypass channel can also be designed in conjunction with the array to maintain constant flux across the device (FIG. 20). In this example, the bypass channel is designed to remove a certain amount of flow so that the flow in the array is not disturbed, ie substantially constant. Such a design can also be used with dual arrays (FIG. 21). In this design, the central bypass channel may be shared by two duplex arrays.

Optimal boundary design If the array is assumed to be infinitely large, the flow turbulence will be equal in all gaps. The flux φ traveling through the gap will be equal, and the side flux in all gaps will be εφ. In practice, this infinite flow pattern is disturbed by the array boundaries. A portion of the array boundary may be designed to create an infinite array flow pattern. The boundary may be of a flow feed type, i.e. the boundary injects fluid into the array, or may be of a flow extraction type, i.e. the boundary extracts fluid from the array.

  A preferable flow extraction boundary gradually increases in width so as to extract εφ (represented by an arrow in FIG. 22) from each gap (d = 24 μm, ε = 1/60) at the boundary. For example, the distance between the array and the side wall gradually increases so that εφ is added from each gap to the boundary. The flow pattern within this array is not affected by the bypass channel due to this boundary design.

  A preferable flow supply boundary is gradually narrower so that εφ is accurately supplied (represented by an arrow in FIG. 23) to each gap (d = 24 μm, ε = 1/60) at the boundary. For example, the distance between the array and the side wall gradually decreases so that εφ is removed from the boundary toward each gap. Again, the flow pattern within this array is not affected by the bypass channel due to this boundary design.

  The width of the flow supply boundary may be the same as or wider than the gap of the array (d = 24 μm, ε = 1/60) (FIG. 24). For example, if the boundary functions as a bypass channel for analyte recovery, a wide boundary may be preferred. A boundary can be used (represented by the arrows in FIG. 24) that supplies a portion of the total flow rate at the boundary to the array and supplies εφ to each gap at the boundary.

FIG. 25 shows a single bypass channel in a dual array (ε = 1/10, d = 8 μm). This bypass channel comprises two flow supply boundaries. Let the flux crossing the broken line 1 of the bypass channel be Φ _bypass . The flow of φ merges into Φ _bypass from the gap toward the left side of the broken line. The shape of the barrier structure at the boundary is adjusted so that the flow rate toward the array becomes εφ in each gap of the boundary. The flux at the broken line 2 is also Φ _bypass .

On-chip fluid resistor deterministic separation for volume determination and stabilization includes fluid resistance to determine and stabilize the flow rate in the array and also to determine the flow rate recovered from the array. A vessel may be used. Shown in FIG. 26 is a schematic diagram of a planar device; samples, such as blood, inflow channels, buffer inflow channels, drainage outflow channels, and product outflow channels are each connected to the array. The inlet and outlet act as fluid resistors. FIG. 26 also shows the corresponding fluid resistance of these different device components.

Determining the flow rate in the array FIGS. 27 and 28 show the flow of the sample and buffer streams in the array and the corresponding flow width when the device is operated at a uniform depth and a constant pressure drop. The flow rate is determined by dividing the pressure loss by the fluid resistance. In the device shown here, I _blood and I _bypass are equivalent, which determines the corresponding flow width of the blood flow and buffer flow in the array.

By controlling the specific resistance in the outflow channel of the determined product and drainage of the recovered fraction, the recovery tolerance of each flow can be adjusted. For example, in the set of schematics shown here, if R _product is greater than R _waste , the concentration may be more concentrated at the expense of potentially increasing loss to the drainage fraction and diluting the drainage. A high product fraction is obtained. Conversely, if R _product is smaller than R _waste , a more dilute and higher yield product fraction is recovered at the expense of potential contamination from the effluent stream.

Flow Stabilization Each inflow channel and outflow channel can be designed so that the pressure loss across the channel is similar to or exceeds the variation in the overall drive pressure. In a typical example, the inflow and outflow pressure losses are 0.001 to 0.99 times the drive pressure.

Multiplexed deterministic array deterministic separation may be achieved using a multiplexed deterministic array. Providing multiple arrays on the device increases sample throughput and allows simultaneous processing of multiple samples or portions of samples for different fractions or manipulations. Multiplexing is also suitable for preparative applications. The simplest multiplex device consists of two devices connected in series, ie in cascade. For example, the outflow from the main flux of one device may be connected to the inflow of a second device. Or alternatively, the outflow from the secondary flux of one device may be connected to the inflow of a second device.

The doubled two arrays may be arranged side by side like a mirror image, for example (FIG. 29). In such an arrangement, the critical size of the two arrays may be the same or different. In addition, the arrays may be arranged so that the main flux flows to the boundaries of the two arrays, the ends of each array, or a combination thereof. Such multiplexed arrays may further include a central region disposed between the arrays, eg, for recovering analytes that exceed a critical size, or for denaturing the sample, eg, by buffer exchange, reaction, or labeling. .

In addition to forming a multiplexed duplex on the device, two or more arrays with separate inflows may be placed on the same device (FIG. 30A). Such an arrangement can be used for multiple samples, or the multiple arrays may be connected to the same inlet for simultaneous processing of the same sample. In simultaneous processing of the same sample, the outlet may or may not be fluidly connected. For example, if multiple arrays have the same critical size, the outlets may be connected to increase sample throughput. As another example, the arrays may not all have the same critical size, or the analytes in the array may not all be treated the same, in which case the outlet is fluidic. It may not be connected to.

  Multiplexing can also be achieved by placing multiple duplex arrays on a single device (FIG. 30B). Multiple arrays, whether double or single, can be arranged in a three-dimensional relationship, such as possible to each other.

Exemplary Multistage Devices In addition to the devices described above, the following exemplary multistage deterministic devices may also be included in the devices of the present invention. For example, FIG. 58A shows a “cascade” arrangement, in which the outlet 1 of one device is connected to the sample inlet of a second device. This allows an initial separation step using the first device so that the sample introduced into the second device is already enriched for the target cells. The critical size of the two devices may be the same or different, depending on the application.

  In FIG. 60, an unlabeled cell sample is introduced from the sample inlet to the first device cascaded, and a buffer containing the labeled reagent is introduced from the fluid inlet to the first device. The epithelial cells are deflected and flow out of the central outlet in a buffer containing the labeling reagent. This concentrated labeled sample is then introduced through the sample inlet into the second device in cascade, while the buffer is added to the second device through the fluid inlet. Further enrichment of the target cells and separation of the free labeling reagent is achieved, and the enriched sample can be further analyzed. Alternatively, the labeling reagent may be added directly to the sample flowing out from the central outlet of the first device before being introduced into the second device. The use of a cascaded arrangement allows a lower amount or higher concentration of labeling reagent to be used at a lower cost compared to the one-stage device arrangement of FIG. 60; The presence of a separation step significantly reduces any non-specific binding that can occur.

  Another option in the arrangement of the stages of two or more devices is a “bandpass” arrangement. This arrangement is shown in FIG. 58B, where the outlet 2 of one device is connected to the sample inlet of the second device. This allows the initial separation step to be performed using the first device so that the sample introduced into the second device comprises cells that have not been deflected within the first device. This method may be useful when the target cell is not the largest cell in the sample; in this case, the first stage is used to deflect large particle size non-target cells to the central outlet. This number can be reduced. Similar to the cascade arrangement, the critical sizes of the two devices may be the same or different, depending on the intended application. For example, different critical sizes are appropriate for applications requiring separation of epithelial cells compared to applications requiring separation of smaller endothelial cells.

  In FIG. 66, the cell sample preincubated with the labeling reagent is introduced into the sample inlet of the first device in the bandpass arrangement, and the buffer is introduced into the first device through the fluid inlet. In the first device, the large particle size non-target cells are deflected and flow out of the central outlet, while the mixture comprising the target cells, small particle size non-target cells, and labeling reagent is the outlet port 2 of the first device. Arranged to flow out of. This mixture is then introduced into the second device through the sample inlet, while buffer is added to the second device from the fluid inlet. Concentration of the target cells and separation of the free labeling reagent is achieved and the concentrated sample can be further analyzed. Non-specific binding of the labeling reagent to the deflected cells in the first stage is tolerated in this method because the deflected cells and any bound labeling reagent are removed from the system.

  In any multi-stage deterministic device arrangement described above, the devices and their connections may be integrated into a single device. For example, a single cascade device with two or more stages is possible, as is a single bandpass device with two or more stages. The multi-stage effluent is then combined with the reservoir inflow.

A small occupied area array deterministic device can be characterized by a small occupied area. Reducing the array footprint reduces costs and reduces the number of analyte collisions with the barrier structure, thereby eliminating potential mechanical damage and other effects to the analyte. The length of the multistage array can be shortened if the boundary between the stages is not perpendicular to the flow direction. Shortening the length becomes important as the number of stages increases. A three-stage array with a small occupation area is shown in FIG.

As described above, the present invention is a device for concentrating analytes such as particles including bacteria, viruses, fungi, cells, cellular components, viruses, nucleic acids, proteins, and protein complexes. And a method. Examples of fluid samples contemplated by the present invention include whole blood, sweat, tears, ear fluid, sputum, lymph fluid, bone marrow suspension, lymph fluid, urine, saliva, semen, vaginal fluid, cerebrospinal fluid, brain fluid, ascites , Secretions of milk, airways, intestines, and urinary tract, and biological fluid samples such as amniotic fluid. In addition, other biological samples that can be lysed or suspended (eg, biopsy samples) are samples contemplated by the systems and methods described herein. In addition to concentration, the device can also be used to perform various operations on the analyte in the sample. Such operations include, for example, denaturing the analyte itself, such as magnetic properties, or denaturing the fluid carrying the analyte. Preferably, the device is used for the concentration of a rare analyte from a heterogeneous mixture, or for the denaturation of a rare analyte, for example by exchanging the liquid in the sample or contacting the analyte with a reagent. Such a device allows a high degree of concentration with limited stress on potentially fragile analytes such as cells, where the device of the present invention does not provide mechanical lysis or intracellular activation of cells. Reduced.

  Although primarily described with respect to cells, the devices of the present invention are based on magnetic properties (or lack thereof) and / or other concentration methods described herein, such as concentration based on hydrodynamic size. Any analyte that can be isolated, concentrated, or removed can be used.

  Deterministic devices and other analytical devices are used, for example, in concentrated samples where analytes are in contact with each other, interact hydraulically, or affect the flow distribution around another analyte. Can also be used. For example, white blood cells can be separated from red blood cells in whole blood from a human donor by a deterministic device. Human blood generally contains up to 45% by volume of cells. As the cells flow through the array, they are in physical contact and / or hydraulically coupled to each other. FIG. 32 schematically illustrates how closely packed cells in the array physically interact with each other.

  As already mentioned, the devices and methods of the present invention may include separating one or more analytes from a sample based on their intrinsic or external magnetic properties. In one embodiment, the sample is treated with a reagent that changes the magnetic properties of the analyte. The modification may be through magnetic particles or through reagents that alter the intrinsic magnetic properties of the analyte. Thus, magnetically responsive analytes are attracted to the device surface and the desired analyte in the sample (eg, rare cells such as fetal cells, pathogenic cells, cancer cells, or bacterial cells) is retained in the device. Can do. In another aspect, the desired analyte is retained in the device by a mechanism based on size, shape, or deformability. In another embodiment, negative sorting is utilized, where undesired magnetically sensitive analytes are bound in the device and the desired analyte is not bound. In addition to this coupling, the path of the analyte that is susceptible to magnetism can be altered by the magnetic field, for example, to deflect the desired or undesired analyte in a particular direction, such as the direction of the outlet. . In either embodiment, a MACS column can be used for retention of analyte (eg, analyte bound to magnetic particles).

  In embodiments of the invention using positive sorting, for example, magnetically coupled into the device, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the analyte of interest. %, 95%, 98%, or 99% is preferably retained in the concentrated sample. The device surface is preferably designed to minimize non-specific binding of non-target analytes. Further, it is preferred that at least 99%, 98%, 95%, 90%, 80%, or 70% of the non-target analyte is not retained in the concentrated sample, for example by not being magnetically coupled into the device. Selective retention in the device allows separation of specific analyte populations from mixtures such as blood, sputum, urine, and soil, air, or water samples.

  With the method and device of the present invention, for example, at least 0.01%, 0.1%, 1%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or 95% of the concentrated sample High purity concentrated samples can be produced such that is the desired analyte. When the analyte is a cell, such as fetal erythrocytes or epithelial cells, it is particularly preferred that the purity be high so that quantitative PCR methods can be used. The devices of the present invention also allow for high purity in high yields (ie, desired analyte retention). For example, at least 90% of the desired analyte present in the sample, such as fetal red blood cells, is retained in the sample concentrated with the device of the present invention, and at least 90% of the undesired analyte, such as white blood cells, is retained. For example, at least 95% or 99.9% is not retained in the concentrated sample.

  In further embodiments, the devices of the present invention comprise bloodborne pathogens, bacterial and viral quantities, isolation and detection of airborne pathogens dissolved or suspended in aqueous media, pathogen detection in the food industry, and chemical And environmental sampling for biological hazards. Furthermore, the magnetic particles may coexist with the capture site and the candidate drug compound. The capture of the subject cell may be further analyzed for interaction with the captured cell and the immobilized drug compound. Thus, for use in high-throughput cell-based secondary screening of candidate compounds in drug discovery processes, the device isolates cell subpopulations from complex mixtures and reacts these cells with candidate drug compounds. Can be used for both analysis. In other embodiments, testing for receptor-ligand interactions in drug discovery localizes capture sites or receptors on magnetic particles to flow through a complex mixture of candidate ligands (or agonists or antagonists). Can be achieved within the device. The ligand of interest is captured and its binding phenomenon can be detected by secondary staining with a fluorescent probe, for example. This aspect makes it possible to quickly identify the absence or presence of a known ligand from a complex mixture extracted from a tissue or cell treatment, or to identify candidate drug compounds.

Selective retention of the magnetic particle analyte is a barrier structure that attaches to a magnetic particle (eg, a barrier structure present in the device or increases the area where the analyte interacts to increase the likelihood of binding. Can be achieved by introducing it into the device of the present invention. By binding the capture site onto the magnetic particle, specific binding with the target analyte can be caused. In another embodiment, the magnetic particles may be arranged so that only analytes of a certain size, shape, or deformability can selectively pass through the device. Combinations of these aspects are also envisioned. For example, a device may be configured to hold a particular analyte based on size and hold other analytes based on binding. In addition, the device may be designed to bind one or more analytes of interest, for example, regions may be arranged in series, parallel, or distributed within the device, or two or more capture sites. Are arranged on the same or adjacent magnetic particles, for example by being coupled to the same barrier structure or region. In addition, multiple capture sites (eg, anti-CD71 and anti-CD36) that have specificity for the same analyte in the device or for the same or different magnetic particles can be combined with, for example, the same or different barrier structures or It can be used by arranging in a region.

  The flow conditions within the device are generally set so that the analyte is processed very gently to prevent analyte damage. Flow from a positive or negative pressure pump or fluid column may be utilized to deliver the analyte into and out of the microfluidic device of the present invention. This device allows for mild processing while maximizing the frequency of collision of each analyte with one or more magnetic particles. The analyte of interest interacts with any capture site upon collision with magnetic particles. The capture site may co-localize with the barrier structure as a result of the attraction by the magnetic field in the designed device. This interaction allows capture and retention of the analyte of interest at a predetermined location. The captured analyte can be released by demagnetizing the magnetic region holding the magnetic particles. For selective release of analyte from the region, demagnetization can be limited to selected barrier structures or regions. For example, the magnetic field may be an electromagnetic field, which makes it possible to arbitrarily apply a magnetic field for each region or barrier structure. In other embodiments, the particles are released by cleaving the bond between the analyte and the capture site, for example, by chemical cleavage, blocking non-covalent interactions, or reducing the magnetic response of the bound analyte. can do. For example, some iron particles are conjugated to monoclonal antibodies via a DNA linker; using a DNA-degrading enzyme, the bond can be cleaved to release the analyte from the iron particles. Alternatively, selective release treatment may be performed using an antibody fragmenting protease (eg, papain). Particularly in the case of the hard magnetic region, the shear force on the magnetic particles may be increased to release the magnetic particles from the magnetic region. In other embodiments, the captured analyte is not released and can be analyzed or further processed in the retained state.

  FIG. 76 shows an example of a reservoir designed to capture and isolate cells expressing transferrin receptor from a complex mixture. Monoclonal antibodies against the CD71 receptor are readily available and commercially available in a covalent bond with magnetic materials such as, but not limited to, iron-doped polystyrene and iron particles or iron colloids (eg, Miltenyi and Dynal). The mAB for CD71 bound to the magnetic particles flows into the reservoir. The antibody-coated particles are attracted to the barrier structure (eg, columnar), bottom, and wall and are retained by the strength of the magnetic field interaction between the particles and the magnetic field. Particles that are between the barriers and particles that are gently held in the area of influence of the localized magnetic field away from the barrier structure are removed by rinsing (the flow velocity is the hydraulic shear stress on the particles away from the barrier structure) Can be adjusted to be greater than the magnetic force).

  FIG. 77 is a preferred embodiment in which a reservoir is applied to capture and release CD71 positive cells of a complex mixture, such as blood, using holotransferrin. Holotransferrin has a high iron content, is commercially available, and has a higher affinity constant and interaction specificity with the CD71 receptor than the corresponding monoclonal antibody. Iron bound to a transferrin ligand plays a dual role of retaining the ligand's conformation for binding to cellular receptors and acting as a molecular paramagnetic element for retaining the ligand on the barrier structure. .

Concentration In one embodiment, the device of the invention is used to make a sample enriched with a desired analyte, for example based at least in part on magnetic properties and optionally on hydrodynamic size. . Such concentration applications include concentration of analytes such as particles comprising rare cells. The device can also be used to concentrate cellular components such as organelles (eg nuclei). In the devices and methods of the invention, at least 1%, 10%, 30%, 50%, 75%, 80%, 90%, 95%, 98%, or 99% of the desired analyte compared to the initial mixture. While the desired analyte is at least 1, 10, 100, 1000, 10,000, 100,000, or 1, for one or more unwanted analytes Preferably it is potentially concentrated up to 000,000 times. Concentration increases the concentration of the desired analyte relative to unwanted analyte in the sample, but may result in dilution of the desired analyte relative to the initial sample. The dilution is preferably 90% or less, for example, 75% or less, 50% or less, 33% or less, 25% or less, 10% or less, or 1% or less.

  In another aspect, the device of the invention is used to make a sample enriched for rare analytes. Generally, a rare analyte is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3% relative to the total analyte in the sample, Less than 2%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, less than 0.00001%, or less than 0.000001% Or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, 1% of the total mass of the sample An analyte that is less than, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, less than 0.00001%, or less than 0.000001%. Typical rare analytes depend on the sample, but are fetal cells, bone marrow cells, progenitor cells, stem cells (eg, undifferentiated stem cells), foam cells, cancer cells, immune system cells (host or graft), epithelial cells , Endothelial cells, endometrial cells, trophoblasts, connective tissue cells, bacteria, fungi, viruses, and pathogens (eg, bacteria or protozoa). Such rare cells can be isolated from body fluids such as blood or samples of environmental sources such as pathogens in water samples, for example. Fetal red blood cells can be enriched from maternal peripheral blood, for example, to determine the sex of the developing fetus and to identify aneuploidy or genetic features, such as mutations. Circulating tumor cells are usually epithelial cell types and derived from epithelial cells, but these cells can also be enriched from peripheral blood for the purpose of diagnosis and treatment progress monitoring. Circulating endothelial cells can be concentrated from peripheral blood as well.

  Body fluids or environmental samples can also be screened for pathogens such as, for example, coliforms, blood-derived diseases such as sepsis, or bacterial or viral meningitis. Rare cells also include cells of one organism that are present in another organism, such as cells of a transplanted organ.

  The amount of blood or other body fluid removed may vary depending on the type of mammal and its condition, for example pregnancy or the stage of the disease such as cancer. In some embodiments, less than 50 mL, less than 40 mL, less than 30 mL, less than 20 mL, less than 10 mL, less than 9 mL, less than 8 mL, less than 7 mL, less than 6 mL, less than 5 mL, less than 4 mL, less than 3 mL, less than 2 mL, less than 1 mL, 0 Less than 5 mL, less than 0.1 mL, less than 0.05 mL, or less than 0.01 mL of body fluid is obtained from an individual. In some embodiments, 1 to 50 mL, 2 to 40 mL, 3 to 30 mL, or 4 to 20 mL of body fluid is obtained from an individual. In other embodiments,> 5 mL,> 10 mL,> 15 mL,> 20 mL,> 15 mL,> 30 mL,> 35 mL,> 40 mL,> 45 mL,> 50 mL,> 55 mL,> 60 mL,> 65 mL,> 70 mL,> 75 mL > 80 mL,> 85 mL,> 90 mL,> 95 mL, or> 100 mL of body fluid is obtained from an individual. For example, in certain embodiments, the systems and methods herein can detect and isolate rare cells (eg, fetal cells) from maternal blood samples of less than 5 mL or less than 3 mL. In other examples, the systems and methods herein can be used to analyze or concentrate rare cells from large volumes of blood, such as greater than 20 mL or greater than 50 mL. Any of the above functions may be, for example, less than 1 day, or less than 12 hours, less than 10 hours, less than 11 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, It can be performed within less than 2 hours, less than 2 hours, or less than 1 hour. The entire collected sample may be applied to the devices herein for enrichment and / or detection of rare cells. Alternatively, the sample may be processed so that only certain components are introduced into the device.

  In addition to concentrating rare analytes, the device can be utilized for preparative applications. Typical preparation applications include the generation of cell packs from blood. In one example, magnetic separation alone or in combination with deterministic enrichment can be designed to produce a fraction enriched in platelets, red blood cells, and white blood cells. By using a multiplexed or multistage device, all three cell fractions can be generated simultaneously or sequentially from the same sample. In other embodiments, the device can be used to separate nucleated cells from anucleated cells, eg, in cord blood samples.

  The device of the present invention is useful in situations where the analyte to be concentrated is susceptible to damage and other degradation. As described herein, the device can be designed to concentrate the analyte with a minimal number of collisions between the analyte (eg, cells) and the barrier structure or other surface. By minimizing the number of collisions in this way, mechanical damage to the analyte (eg cells) can be reduced, and in the case of cells, intracellular activation caused by collisions can also be prevented or reduced. Gentle treatment preserves a limited number of rare analytes in the sample, prevents or reduces ruptures that cause contamination or degradation by intracellular components in the case of cells, and maturation and activity of cells such as stem cells and platelets To prevent or reduce In preferred embodiments, damaged (eg, activated or mechanical lysis) analytes are less than 30%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% Concentration is performed so that

  Shown in FIG. 33 is a general size distribution of cells in human peripheral blood. White blood cells range from 4 μm or less to 18 μm or less, while red blood cells are 1.5 μm or less (short diameter). Deterministic devices designed to separate white blood cells from red blood cells typically have a cut-off size of 2-4 μm and a passage size greater than 18 μm. Such a device may be used in combination with magnetic separation as described herein.

  FIG. 57A shows the operation of a deterministic device for concentration. Cell samples are added from the sample inlet and buffer is added from the fluid inlet. Cells that are smaller than the critical size move through the device without being deflected and are contained in the original sample medium and discharged from the end outlet. Cells larger than the critical size, such as epithelial cells, are deflected and contained in a buffer added from the fluid inlet and discharged from the central outlet. Therefore, by manipulating this device, a sample enriched with larger and smaller cells is produced. Since epithelial cells are the largest class of cells in the bloodstream, the size and geometry of the device gaps allow samples that are substantially enriched for epithelial cells to pass from the central outlet through a single pass through the device. While generating, it can be selected to direct virtually all other cell types to the end outlet.

  As shown in FIG. 57A, the deterministic devices included in the present invention need not be dual in order to perform operations as described herein. The schematic shown in FIG. 57B can represent a dual device or a single array.

  By combining magnetic separation with deterministic separation, enrichment can be facilitated in various ways. For example, the analyte of interest (eg, a cell) is labeled with a bead (eg, an immunoaffinity bead), thereby increasing its size (as shown in FIG. 59), and potentially increasing the magnetic properties of the analyte. It can also be changed. In the case of epithelial cells, this further increases the size, resulting in a further increase in separation efficiency. Or as an alternative, for smaller analytes (eg cells), to the extent that the analyte is the largest object in the sample or occupies a different size range compared to other components of the sample, or The size may be increased to co-purify with other analytes. The beads can be made from polystyrene, magnetic material, or any other material that can adhere to an analyte (eg, a cell). Such beads preferably have an intermediate buoyancy that does not interfere with the flow of labeled cells within the device. Changes in the size of the analyte can be utilized in enrichment based on magnetic and deterministic properties used in parallel or series arrangements.

Denaturation In other embodiments, the analyte of interest may be contacted with a denaturant capable of chemically or physically denaturing the analyte or fluid in the sample with or without concentration. Applications include purification, buffer exchange, labeling (eg, immunohistochemical, magnetic, and histochemical labeling, cell staining, and flow in-situ fluorescence hybridization (FISH)), Examples include magnetic denaturation, cell fixation, cell stabilization, cell lysis, and cell activation.

  By such a method, the analyte can be transferred from the sample into another liquid (eg, buffer exchange). Figure 34A schematically illustrates this effect for a single stage deterministic device, Figure 34B illustrates this effect for a multistage deterministic device, and Figure 34C illustrates this effect deterministically. FIG. 34D illustrates this effect for a multi-stage dual array of deterministic devices. Similarly, magnetic separation can be used to hold the analyte in the device or to deflect the buffer in the desired direction for buffer exchange. By using such methods, analytes (eg, blood cells) can be concentrated in the sample. Such transfer of analyte from one liquid to another can be used to perform a series of denaturations, such as light staining blood on-chip. Such a series of denaturations may involve reacting the analyte with a first reagent, then transferring the analyte to a wash buffer and reacting with another reagent.

  Figures 35A to 35C show further examples of modifications in a two-stage deterministic device with two bypass channels. In this example, a larger analyte is transferred from the blood to a buffer (eg, comprising a reagent that changes the magnetic properties of the analyte) and collected at stage 1. Analytes with intermediate sizes are transferred from blood to buffer (eg, with reagents that change the magnetic properties of the analyte) at stage 2, and smaller analytes that do not move from blood to buffer at stage are also collected. . FIG. 35B shows the size cutoff in the two stages, and FIG. 35C shows the size distribution of the three fractions collected. The collected fraction can subsequently be subjected to a magnetic-based concentration treatment.

  FIG. 36 shows an example of a degeneration, such as magnetic properties, in a two-stage deterministic device having a bypass channel located between the lateral end of the array and the channel wall. FIG. 37 shows a deterministic device similar to FIG. 36 except that the two stages are connected by a fluidic channel. FIG. 38 shows denaturation in a deterministic device having two stages with a small footprint. Figures 39A-39B show denaturation in a device where effluent from the first and second stages is captured in one channel. FIG. 40 shows another device for use in the method of the present invention.

  FIG. 41 illustrates the use of a deterministic device to perform multiple sequential denaturations on an analyte. In this device, the analyte is transferred from the sample into a reagent that reacts with the analyte, and the denatured analyte is then transferred into a buffer, thereby removing unreacted reagents or reaction byproducts. Additional steps may be added (eg, steps described herein).

  Concentration and denaturation can be combined. For example, the desired cells can be contacted with a lysis reagent, and cellular components such as nuclei are enriched based on size, magnetic properties, or both. In another example, the analyte can be contacted with a particulate labeling agent such as magnetic beads that binds the analyte. Unbound particulate labeling agent can be removed based on size, magnetic properties, or both.

Concentration The device of the present invention can also be used to concentrate a sample of interest, such as cells. In one example shown in FIG. 62, a cell sample is introduced into the sample inlet of the deterministic device. Lowering the volume of the buffer introduced into the fluid inlet so that it is significantly lower than the volume of the cell sample results in an increase in the concentration of the target cells in a smaller volume. Similarly, retention of magnetically responsive analyte in the channel can be utilized to concentrate the analyte, for example, by releasing the retained analyte into a smaller volume. This concentration step can improve the results of the analysis performed in the downstream step.

Cell Lysis The device of the present invention can also be used for cell lysis. To accomplish this in a deterministic device, follow a procedure similar to the protocol used in enrichment: add cell sample from the sample inlet of the device (FIG. 63) and add lysis buffer from the fluid inlet. . As mentioned above, cells larger than the critical size are deflected into the lysis buffer and these cells are lysed. As a result, the sample discharged from the central outlet contains lysed cellular components comprising organelles, while the entire unbiased cell is discharged from the other outlet. Similarly, cells retained (or not retained) in the device based on magnetic properties can be contacted with a lysis reagent to release, for example, intracellular components of the magnetically bound analyte. Thus, this device provides a method of selectively lysing target cells.

Concentrated analytes such as downstream analysis, eg, rare cells and / or components, may be detected using any means known in the art. For example, in certain embodiments, the detection module herein can be, for example, a microscope, camera, spectrometer, or hyperspectral imager (eg, Vo-Dinh et al., IEEE Eng. Med. Biol. Mag., Vol. 23, p. 40). -49, (2004)). Detection may comprise the use of selective staining indicating the presence or absence of the analyte of interest and the detection of a color change. In certain embodiments, cell identification by staining uses a non-direct immunostaining method that uses an unlabeled primary antibody followed by an enzyme-conjugated secondary antibody. Typical enzymes include horseradish peroxidase and alkaline phosphatase. Dye staining occurs with known colorless substrates for the complex enzyme. In certain embodiments, the rare cells that are stained and identified are further analyzed for chromosomal abnormalities by nucleic acid hybridization using specific probes. This triage strategy preferably uses staining for rare cells and different markers for nucleic acid probes.

  An important requirement for many diagnostic tests is to remove free or unreacted denaturant from the analytical sample or reduce it to below acceptable levels. In one embodiment, the reagent is a labeling reagent. As described above, the device of the present invention can separate free labeling reagent from labeling reagent bound to an analyte (eg, a cell). Therefore, it is possible to perform bulk measurements of reaction samples without receiving a significant level of background interference from free labeling reagents. In one example, fluorescent antibodies that are selective for specific epithelial cell markers such as EpCAM are used. The fluorescent moiety may include a Cy dye, Alexa dye, or other fluorophore-containing molecule. The resulting labeled sample is then analyzed by measuring the fluorescence of the resulting sample of analyte concentrated using a label such as a cell with a fluorometer. Alternatively, a chromophore-containing label may be used with a fluorometer. Using the obtained measured value, the number of target analytes such as cells in the sample can be quantified.

  Many other measurement methods and labeling reagents are also useful in the methods and devices of the present invention. A labeled antibody may have a covalently linked enzyme that cleaves the substrate and changes the absorbance at a certain wavelength. The degree of cleavage is quantified with a spectrometer. Depending on the substrate used, colorimetric or luminescent readings are possible. Beneficially, the measurement signal can be greatly amplified by using an enzyme label, and the detection limit can be lowered.

  Quantum dots, such as Quantum Dot Corp. Qdots (registered trademark) can be used as a labeling reagent that covalently binds to a capture site such as an antibody. Qdots is resistant to photobleaching and can also be used in combination with two-photon excitation measurements.

  Another possible labeling reagent useful in the methods of the present invention is phage. The phage display method is a technique in which a binding peptide is displayed by a recombinant phage system having a strong binding affinity for a target such as a protein found on the surface of a target cell. A peptide sequence corresponding to any phage is encoded in the nucleic acid of the phage. Therefore, phages are potentially small compared to analytes such as cells and are therefore easy to separate, and also retain nucleic acids that can be analyzed and quantified by PCR or similar techniques, so the presence of cells present in a concentrated sample Since the number can be quantified, it is a useful labeling reagent.

  FIG. 65 represents the use of phage as a labeling reagent, where the two stages of the deterministic device are arranged in cascade. The method shown in FIG. 65 corresponds to FIG. 64 generally shown except for the labeling reagent to be used. Similarly, magnetic concentration may be used.

  Downstream analysis may include accurate quantification of the number of desired analytes (eg, cells) in the sample being analyzed. In order to obtain accurate quantitative results, generally the amount of target of the labeling reagent (eg the surface antigen of the subject cell) must be known or predictable (eg based on the level of expression in the cell) It is also necessary that the binding of the labeling reagent proceed in a predictable manner, for example in the absence of interfering substances. Thus, a device or method that produces a highly concentrated cell sample prior to introduction of a labeling reagent is particularly useful. Furthermore, when the desired analyte is scarce (eg, epithelial cells in a blood sample), a labeling reagent that amplifies the generated signal is preferred. Examples of reagents for amplifying signals include enzymes and phages. Other labeling reagents such as quantum dots that do not allow simple signal amplification but generate strong signals are also preferred. Quantification can also be performed with non-denaturing or unlabeled analytes.

  If the devices and methods of the invention are used to enrich cells in a sample, further quantification and molecular biological analysis can be performed on the same set of cells. The gentle treatment of cells by the device of the present invention, in combination with the previously described bulk measurement method, maintains the integrity of the cells so that further analysis is possible if desired. For example, following bulk measurements, techniques that disrupt cell integrity can be performed; such techniques include DNA or RNA analysis, proteome analysis, or metabolome analysis. An example of such an analysis is the PCR method, in which cells are lysed and the concentration of a specific DNA sequence is amplified. Such a technique is particularly useful when the number of isolated target cells is very small.

Epithelial cells were detached from the diagnostic solid tumor cancer, breast cancer, colon cancer, are found in liver cancer, ovarian cancer, prostate cancer, and circulatory system of a patient's lung cancer. In general, the presence of circulating tumor cells (CTC) after treatment is associated with tumor progression and spread, poor therapeutic efficacy, disease recurrence, and / or decreased survival. Thus, counting CTCs provides a means of stratifying patients for features that serve as a basis for predicting initial risk and subsequent risk based on therapeutic effects.

  Unlike tumor-derived cells in the bone marrow, which are dormant and have a long life span, CTC, which is an epithelial cell type and derived from epithelial cells, has a short half-life of about 1 day, and its presence has recently been influxed from proliferating tumors. (Patel et al., Ann. Surg., Vol. 235, p. 226-231, 2002). Thus, the CTC can reflect the current clinical state of the patient's disease and therapeutic effects. CTC enumeration and characterization has potential value in assessing cancer prognosis and in monitoring therapeutic effects for early detection of treatment failures that can lead to disease recurrence. In addition, CTC analysis allows early detection of recurrence in patients with prodromal symptoms who have completed a series of treatments. Currently, people without measurable disease are not eligible to participate in clinical trials of promising new treatments (Braun et al., N. Engl. J. Med., Vol. 351, p. 824-826). 2004).

  The devices and methods of the present invention can be used to evaluate cancer patients and patients at risk for cancer. For example, a blood sample is taken from a patient and introduced into the device of the present invention to separate epithelial cells from other blood cells. The number of epithelial cells in the blood sample is quantified using, for example, the methods described herein. For example, the cells can be labeled with an antibody that binds EpCAM, which antibody can have a covalently bound fluorescent label or can be bound to magnetic particles. The concentrated sample made with this device can then be subjected to a bulk measurement, from which the number of epithelial cells present in the original sample of blood can be quantified. Microscopic techniques can be used to quantitate cells visually and correlate the number of corresponding labeled cells in the blood sample with the bulk measurement.

  By taking a series of measurements over days, weeks, months, or years, the level of epithelial cells present in the patient's bloodstream can be tracked as a function of time. For patients who already have cancer, this method provides useful information indicating disease progression and appropriate treatment based on the increase, decrease, or no change in circulating epithelial cells in the patient's bloodstream. Help the doctor in choosing a law. For patients at risk of cancer, a sudden increase in the number of detected cells can provide an early warning that the patient has developed a tumor. This early diagnosis, when combined with subsequent treatments, tends to result in improved patient prognosis compared to the absence of this diagnostic information.

  Diagnostic methods include bulk measurements of labeled epithelial cells isolated from blood as well as techniques that disrupt cell integrity. For example, PCR is performed on a sample with a very small number of isolated target cells; information on the type of tumor originating from the analyzed cells by using primers specific for a particular cancer marker Can be obtained. RNA analysis, proteome analysis, or metabolome analysis can be additionally performed as means for diagnosing the type of cancer that the patient has.

  One important diagnostic indicator of lung cancer and other cancers is the presence or absence of certain mutations in the epidermal growth factor receptor (EGFR). EGFR consists of an extracellular ligand binding domain, a transmembrane region, and an intracellular tyrosine kinase (TK) domain. The normal physiological role of EGFR is to bind downstream ErbB ligands, including epidermal growth factor (EGF), at the extracellular binding site and cause downstream intracellular signals that cause proliferation, survival, motility, and other related activities. Inducing a cascade. Many non-small cell lung tumors with EGFR mutations respond to small molecule EGFR inhibitors such as gefitinib (Iressa, manufactured by AstraZeneca), but in many cases eventually develop secondary mutations that cause drug resistance. As shown. By using the devices and methods of the present invention, patient monitoring is performed by frequently collecting blood samples for patients taking such drugs and quantifying the number of epithelial cells in each sample as a function of time. It can be performed. This provides information on the course of the disease. For example, a decrease in the number of circulating epithelial cells over time suggests that the severity of the disease has been alleviated and the tumor has become smaller. Immediately after quantification of epithelial cells, these cells can be analyzed by PCR to identify what mutations may be present in the EGFR gene expressed in the epithelial cells. It is known that certain mutations, such as those concentrated around the ATP binding pocket in the EGFR TK domain, increase the sensitivity of cancer cells to gefitinib inhibition. Therefore, the presence of such mutations supports the diagnosis that the cancer shows a favorable response to treatment with gefitinib. However, many patients who respond favorably to gefitinib eventually develop a secondary mutation, often a methionine to threonine substitution at position 790 in exon 20 of the TK domain. This replacement makes the cells resistant to gefitinib. Testing for this mutation can also be performed by using the devices and methods of the present invention, which provides additional diagnostic information regarding the course of the disease and the likelihood of responding to gefitinib or similar compounds.

Detection of Fetal Cells The devices and methods described herein can be used, for example, to screen fetal conditions or abnormalities on blood samples taken from pregnant humans. When screening fetuses, blood samples from pregnant mammals or humans whose gestation period is within 24 weeks, within 20 weeks, within 16 weeks, within 12 weeks, within 10 weeks, within 8 weeks, or within 4 weeks are taken. Can be collected. In other embodiments, fetal cell screening and detection may be performed after termination of pregnancy.

  For example, in certain embodiments, rare cells are detected by staining for antigens such as ε / γ globin (cytoplasm), GPA, i-antigen, CD71, or combinations thereof. The combination of ε globin and γ globin is found in 95 to 100% of fetal nucleated red blood cells (fNRBC) from 10 to 24 weeks of gestation. Al-Mufti et al., Haematologica, Vol. 85, p. 357-362 (2001), Choolani et al., Mol. Hum. Reprod. Vol. 9, p. 227-235 (2003). This ε-γ combination or γ globin alone is described in, for example, Bohmer, Br. J. et al. Haematol. Vol. 103, p. 351-60 (1998), Choolani et al. (2003), Christensen et al., Fetal Dian. Ther. Vol. 20, p. 106-112 (2005), and Hennerbichler et al., Cytometry, Vol. 48, p. 87-92 (2002), which has been used for staining of fNRBC. Globin is a selection criterion with high specificity (over 10,000 times), and the number of false detections per fNRBC was less than 10 with or without CD71 enrichment. Antibodies to both globins are known to those skilled in the art. The result of staining can be generated as a binary evaluation of positive or negative, or as various intensities indicating the amount of antigen in the analyte.

  Glycophorin A and CD71 are additional antigens that can be used to detect cell types. GPA is present throughout the erythroid lineage. Therefore, it can be used to identify nucleated red blood cells regardless of their maturity level. GPA is thought to be present only on erythroid lineage cells, is usually rarely found on circulating cells, and its abundance increases during pregnancy. For example, Price et al., Am. J. et al. Obstet. Gynecol. Vol. 165, p. 1713-1717 (1991), and Sohda et al., Pregnat. Diagn. Vol. 17, p. 743-752 (1997), FACS sorting showed that the combination of CD71 and GPA was present in at least 0.15% of mononuclear cells during pregnancy.

  For example, Sital et al., Exp. Cell Res. Vol. 302, p. 153-161 (2005), antigen i can also be used as a marker for isolation and / or detection of fetal cells. The i antigen was first published in the 1950s by using patient polyclonal sera. Subsequent data indicated that two forms of this antigen, antigen “I” and antigen “i”, are expressed on adult cells and fetal cells, respectively.

  Once the fetal cells or components of interest are detected, they can be further analyzed for a variety of purposes such as gender, genetic status, and the like. In some embodiments, analysis of fetal cells or components thereof is used to determine the presence or absence of genetic abnormalities such as chromosomal abnormalities, DNA abnormalities, or RNA abnormalities. Examples of autosomal abnormalities include Angelman syndrome (15q11.2-q13), cat cry syndrome (5p-), DiGeorge syndrome and palatal and facial syndrome (22q11.2), Miller-Dieker syndrome (17p13.3). Prader-Willi syndrome (15q11.2-q13), retinoblastoma (13q14), Smith-Magenis syndrome (17p11.2), chromosome 13 trisomy, chromosome 16 trisomy, chromosome 18 trisomy, chromosome 21 trisomy (Down's syndrome), triploid, Williams syndrome (7q11.23), and Wolf Hirschhorn syndrome (4p-), but are not limited to these. Examples of sex chromosome abnormalities include Kalman syndrome (Xp22.3), sulfated steroid deficiency (STS) (Xp22.3), X-chromosome ichthyosis (Xp22.3), Kleinfelter syndrome (XXY), fragile X syndrome, Turner syndrome, super female or X chromosome trisomy, and X chromosome monosomy include, but are not limited to.

  Other less common chromosomal abnormalities that can be analyzed by the system of the invention include deletions (small portions lost), microdeletions (loss of trace material that can contain only a single gene), translocations ( Part of the chromosome is added to another chromosome), and inversion (a section of the chromosome is cut out and reinserted upside down), but is not limited to these.

  In some embodiments, analysis of fetal cells or components thereof is utilized for SNP analysis and prediction of fetal status based on such SNPs.

  In any of the embodiments herein, detection / analysis may be performed using any means known in the art. Methods for detecting / analyzing the gene state include karyotype analysis, in situ hybridization (ISH) (eg, fluorescence in situ hybridization (FISH), dye-colored in situ hybridization) ( CISH), gold nanoparticles in situ hybridization (NISH)), restriction fragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR) method, flow cytometry, electron microscopy, quantum dots, and Examples include, but are not limited to, single nucleotide polymorphisms (SNPs) or nucleic acid arrays for detection of RNA levels. In some embodiments, two or more methods are performed for genetic abnormality detection. For example, a plurality of types of FISH probes or other DNA probes can be used for analysis of a single target cell or component.

Cell Subpopulation Enrichment The methods and devices of the present invention can also be used to enrich a subpopulation of cells. For example, nucleated red blood cells change in morphology and hemoglobin content during maturation. Based on the difference in hemoglobin content, late-stage nucleated cells such as orthochromatic erythroblasts can be separated from early nucleated cells such as polychromatic erythroblasts. FIG. 99 shows the nRBCs at various stages of maturity obtained using the methods described herein.

Erythrocyte phagocytosis Using the methods and devices of the present invention, cells that have absorbed red blood cells (erythrocyte phagocytosis) can also be concentrated. FIG. 100 shows monocytes that have incorporated RBCs and are retained within the magnetic device of the present invention. Erythrocyte phagocytosis is associated with various diseases and genetic abnormalities such as familial hemophagocytic histiocytosis, acute monocytic leukemia, and lymphoma. Detection of these cells can be used to diagnose a particular condition or determine the effectiveness of a treatment. For example, the method of the present invention can be used to quantify the number of cells that have incorporated RBCs therein.

  This method can generally be used for any cell that incorporates or binds a magnetic element such as a magnetotactic bacterium such as Aquaspirillum magnetotoacticum. The magnetotactic bacteria may be further phagocytosed by other cells, in which case these cells can also be enriched using the method of the present invention.

Elevated circulating nRBC levels in nucleated red blood cells are associated with various diseases and genetic abnormalities such as erythroleukemia, severe β thalassemia, Bart's hemoglobin, and immunohemolytic anemia. NRBC or its nuclei can be concentrated by a combination of magnetic concentration and concentration or size based dissolution. Detection of these cells can be used to diagnose a particular condition or determine the effectiveness of a treatment.

Combination of Concentration Methods Any of the concentration methods described herein can be used sequentially or simultaneously to concentrate a target analyte such as a cell or nucleus. When used in combination, they can generally be performed in any desired order. For example, methods include a combination of size separation, magnetic separation, and whole cell lysis or selective lysis. As described herein, the method may further comprise affinity enrichment based on binding to capture sites such as antibodies, or enrichment based on spectroscopic or other measurable properties of the cell or particle. .

  Devices for achieving such a combination of enrichment methods can include independent modules for each enrichment, and two or more enrichments can be a single module or device or, for example, as appropriate Can occur in various types of concentrating devices connected by simple fluidics or relying on manual or robotic movement of samples between devices.

Sample Preparation In the methods described herein, a sample can be used in a treated or untreated state, such as by stabilization or removal of certain components. In one embodiment, the sample is enriched for the analyte of interest, such as cells, before being introduced into the device of the present invention. Methods for concentrating cell populations are described herein and are known in the art, such as affinity mechanisms, magnetic properties, aggregation, and enrichment based on size, shape, and deformability. It is done. Some samples may be diluted or concentrated prior to introduction into the device.

  For example, blood samples are collected within 1 week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, within 1 day, within 12 hours, within 6 hours, within 3 hours, It is preferably applied to the system of the present invention within 2 hours or within 1 hour. In some embodiments, a blood sample is applied to the system of the present invention as soon as it is taken from the patient. The sample is preferably applied to the system of the present invention at 4 ° C to 37 ° C.

  In one embodiment, a reagent is added to the sample to selectively or non-selectively increase the hydraulic size of the analyte within the sample. This modified sample is then inserted, for example, inside a deterministic device. Because the particles are swollen and have increased hydrodynamic size, it is possible to use deterministic devices with larger gap sizes that are easier to manufacture. In a preferred embodiment, the swelling step and the size-based concentration step are performed in an integrated manner on the deterministic device. Suitable reagents include any hypotonic solution, such as deionized water, 2% sugar solution, or pure non-aqueous solvent. Other reagents include beads that bind selectively (eg, by antibodies or avidin-biotin) or non-selectively, such as magnetic beads or polymer beads.

  In another embodiment, a reagent is added to the sample to selectively or non-selectively reduce the hydrodynamic size of the particles in the sample. By non-uniformly reducing the size of the particles in the sample, the hydraulic size difference between the particles increases. For example, nucleated cells are separated from enucleated cells by contracting the cells using high osmotic pressure. Enucleated cells can shrink very small, whereas nucleated cells cannot shrink below the size of the nucleus. Typical contraction reagents include hyperosmotic solutions.

  In another alternative embodiment, the affinityd beads are used to increase the hydraulic size of the analyte of interest relative to other analytes in the sample, thereby making it larger and easier to manufacture. It is possible to use a deterministic device with a gap size.

  Such a change in size can occur in sequence or in parallel with the magnetic concentration, as described herein.

  For example, when collecting a sample such as blood, the sample may be contained in a container containing one or more of the following reagents: stabilizer, preservative, fixative, solubilizer, diluent, Apoptotic agents, anticoagulants, antithrombotic agents, buffers, osmotic pressure adjusting agents, pH adjusting agents, reagents that change magnetic properties, and / or cross-linking agents.

  The fluid can be flowed through the device either actively or passively. The fluid can be delivered by an electric field, centrifugal field, pressure driven fluid flow, electroosmotic flow, or capillary action. In a preferred embodiment, the mean direction of such a field is parallel to the channel wall.

  Any of the following exemplary deterministic devices and methods can be incorporated into the devices of the present invention.

Example
Silicon Device Multiplexing Three-Stage Double Array of Example 1.14 FIGS. 42A to 42E show typical devices having the following characteristics.

Dimensions: 90mm x 34mm x 1mm
Array design: Three-stage type. The gap sizes are 18, 12, and 8 μm for the first, second, and third stages, respectively. The branching ratio is 1/10. Double array structure of single bypass channel.
Device design: 14 double arrays multiplexed, and fluid resistors for flow stability.
Device fabrication: Arrays and channels were fabricated from silicon using standard photolithography and deep silicon reactive etching techniques. The etching depth is 150 μm. KOH wet etching is used to form a through hole for flowing a fluid. The silicon substrate was sealed on the etched surface with a blood compatible pressure sensitive adhesive (9795, 3M, St. Paul, Minn.) To form a capped fluid channel.
Device implementation: The device was mechanically connected to a plastic manifold with an external fluid reservoir to extract blood and buffer supplied to the device to extract the generated fraction.
Device operation: An external pressure source was used to apply a pressure of 2.4 PSI to the buffer and blood reservoirs to regulate fluid delivery and extraction from the mounted device.

Experimental conditions:
Human blood from an agreed adult donor was collected in a K ₂ EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ). Undiluted blood was processed at room temperature within 9 hours after collection using the typical device described above (FIG. 42F). Blood nucleated cells are separated from enucleated cells (erythrocytes and platelets) and plasma, comprising 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St. Louis, MO), calcium and magnesium In a buffer stream of Dulbecco's phosphate buffer (14190-144, manufactured by Invitrogen, Carlsbad, Calif.).

Measuring method:
The total blood count was measured using a Coulter impedance blood analysis device (COULTER (registered trademark) Ac. T diff (trademark), manufactured by Beckman Coulter, Fullerton, Calif.).

result:
43A to 43F show typical samples obtained by the blood analysis device with respect to the blood sample and the drainage fraction (buffer, plasma, red blood cells, platelets) and product fraction (buffer, nucleated cells) obtained by the device. A typical histogram is shown. Table 1 shows the results for five different blood samples.

Table 1

Example 2.14 Silicon Device Multiplexing Single Stage Double Array FIGS. 44A to 44D show typical devices having the following characteristics.

Dimensions: 90mm x 34mm x 1mm
Array design: One stage. The gap size is 24 μm. The branching ratio is 1/60. Double array structure of double bypass channel.
Device design: 14 double arrays multiplexed, and fluid resistors for flow stability.
Device fabrication: Arrays and channels were fabricated from silicon using standard photolithography and deep silicon reactive etching techniques. The etching depth is 150 μm. KOH wet etching is used to form a through hole for flowing a fluid. The silicon substrate was sealed on the etched surface with a blood compatible pressure sensitive adhesive (9795, 3M, St. Paul, Minn.) To form a capped fluid channel.
Device implementation: The device was mechanically connected to a plastic manifold with an external fluid reservoir to supply blood and buffer to the device and to extract the generated fraction.
Device operation: An external pressure source was used to apply a pressure of 2.4 PSI to the buffer and blood reservoirs to regulate fluid delivery and extraction from the mounted device.

Experimental conditions:
Human blood from an agreed adult donor was collected in a K ₂ EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ). Undiluted blood was processed at room temperature within 9 hours after collection using the typical device described above. Blood nucleated cells are separated from enucleated cells (erythrocytes and platelets) and plasma, comprising 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St. Louis, MO), calcium and magnesium In a buffer stream of Dulbecco's phosphate buffer (14190-144, manufactured by Invitrogen, Carlsbad, Calif.).

Measuring method:
The total blood count was measured using a Coulter impedance blood analysis device (COULTER (registered trademark) Ac. T diff (trademark), manufactured by Beckman Coulter, Fullerton, Calif.).

result:
The device was operated at 17 mL / h and achieved greater than 99% erythrocyte removal rate, greater than 95% nucleated cell retention, and greater than 98% platelet removal rate.

Example 3 FIG. Isolation of Fetal Cord Blood FIG. 45 is a schematic diagram of a device used to isolate nucleated cells from fetal cord blood.

Dimensions: 100mm x 28mm x 1mm
Array design: Three-stage type. The gap sizes are 18, 12, and 8 μm for the first, second, and third stages, respectively. The branching ratio is 1/10. Double array structure of single bypass channel.
Device design: 10 duplex arrays multiplexed, and fluid resistors for flow stability.
Device fabrication: Arrays and channels were fabricated from silicon using standard photolithography and deep silicon reactive etching techniques. The etching depth is 140 μm. KOH wet etching is used to form a through hole for flowing a fluid. The silicon substrate was sealed on the etched surface with a blood compatible pressure sensitive adhesive (9795, 3M, St. Paul, Minn.) To form a capped fluid channel.
Device implementation: The device was mechanically connected to a plastic manifold with an external fluid reservoir to supply blood and buffer to the device and to extract the generated fraction.
Device operation: An external pressure source was used to apply a 2.0 PSI pressure to the buffer and blood reservoirs to regulate fluid delivery and extraction from the implemented device.

Experimental conditions:
Human fetal umbilical cord blood was collected into phosphate buffer containing citrate dextrose anticoagulant. 1 mL of blood was processed at room temperature within 48 hours after collection at 3 mL / h using the device described above. Blood nucleated cells are separated from enucleated cells (erythrocytes and platelets) and plasma, 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St. Louis, Mo.) and 2 mM EDTA (15575). -020, calcium and magnesium free Dulbecco's phosphate buffer (14190-144, Invitrogen, Carlsbad, Calif.), Including calcium, including Invitrogen, Carlsbad, Calif.).

Measuring method:
Cell smear samples (FIGS. 46A to 46B) of product fraction and drainage fraction were prepared and subjected to denatured light Giemsa staining (WG16, Sigma-Aldrich, St. Louis, MO).

result:
Fetal nucleated red blood cells were observed in the product fraction (FIG. 46A) and not in the drainage fraction (FIG. 46B).

Example 4
Isolation of fetal cells from maternal blood To demonstrate the feasibility of isolating fetal cells from maternal blood, the devices and steps detailed in Example 1 were used in combination with an immunomagnetic affinity enrichment method. .

Experimental conditions:
Blood was collected in a K ₂ EDTA vacutainer (366643, Becton Dickinson, Franklin Lakes, NJ) from a pregnant pregnant woman who had conceived a male fetus immediately after the abortion. Undiluted blood was processed at room temperature within 9 hours after collection using the device described in Example 1. Blood nucleated cells are separated from enucleated cells (erythrocytes and platelets) and plasma, comprising 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St. Louis, MO), calcium and magnesium In a buffer stream of Dulbecco's phosphate buffer (14190-144, manufactured by Invitrogen, Carlsbad, Calif.). Subsequently, the nucleated cell fraction was labeled with anti-CD71 microbeads (130-046-201, manufactured by Miltenyi Biotech Inc., Oban, Calif.), And MiniMACS ™ MS column (130-042-201, Miltenyi). Biotech Inc., Oban, Calif.) And concentrated according to manufacturer's specifications. Finally, a CD71 positive fraction was dropped on a slide glass.

Measuring method:
The slide glass into which the sample was dropped was stained by a fluorescence in situ hybridization (FISH) method using a Vysis probe (Abbott Laboratories, Downers Grove, Ill.) According to the manufacturer's specifications. Samples were stained for the presence of X and Y chromosomes. In one case, chromosome 21 was also stained with a sample prepared from a pregnant woman known to be chromosome 21 trisomy.

result:
Since male cells are surely present in the CD71 positive population prepared from the nucleated cell fraction, fetal cell isolation was confirmed (FIG. 47). In one abnormal case tested, the pathology of chromosome 21 trisomy was also recognized (FIG. 48).

  Examples 5 to 10 show specific embodiments of the device. In the description of each device, the number of consecutive stages, the gap size of each stage, ε (flow angle), and the number of channels per device (array / chip) are shown. Each device was fabricated from silicon by the DRIE method and each had a thermal oxide layer.

Example 5
This device has five stages in one array.

Array design: Five-stage, asymmetric array.
Gap size:
First stage: 8 μm
Second stage: 10 μm
Third stage: 12 μm
Fourth stage: 14 μm
5th stage: 16 μm
Flow angle: 1/10
Array / chip: 1

Example 6
The device comprises a stage that is a dual array with a bypass channel. The height of the device was 125 μm.

Array design: A symmetric three-stage array with a central collection channel.
Gap size:
First stage: 8 μm
Second stage: 12 μm
Third stage: 18 μm
4th stage:
5th stage:
Flow angle: 1/10
Array / chip: 1
Other: Central collection channel

  FIG. 49A shows the mask used in making this device. 49B-49D are enlarged views of the portion of the mask that defines the inlet, array, and outlet. 50A to 50G are SEM photographs of actual devices.

Example 7
The device includes a stage that is a dual array with a bypass channel. The “fin” was designed to be adjacent to the bypass channel to prevent fluid from the bypass channel from re-entering the array. The chip further included an on-chip fluid resistor. That is, the inlet and outlet had greater fluid resistance than the array. The height of the device was 117 μm.

Array design: Three-stage symmetrical array.
Gap size:
First stage: 8 μm
Second stage: 12 μm
Third stage: 18 μm
4th stage:
5th stage:
Flow angle: 1/10
Array / chip: 10
Other: Central collection channel with large fins, on-chip fluid resistor

  FIG. 51A shows the mask used in making this device. 51B to 51D are enlarged views of the portion of the mask that defines the inlet, the array, and the outlet. 52A to 52F are SEM photographs of actual devices.

Example 8
The device comprises a stage that is a dual array with a bypass channel. The “fin” was designed to be adjacent to the bypass channel to prevent fluid from the bypass channel from re-entering the array. The edge of the fin closest to the array was designed to approximate the array shape. The chip further included an on-chip fluid resistor. That is, the inlet and outlet had greater fluid resistance than the array. The height of the device was 138 μm.

  FIG. 45A shows the mask used in making this device. 45B to 45D are enlarged views of the portion of the mask that defines the inlet, array, and outlet. 53A to 53F are SEM photographs of actual devices.

Example 9
The device comprises a stage that is a dual array with a bypass channel. The “fin” was optimized using Femlab to be adjacent to the bypass channel to prevent fluid from the bypass channel from re-entering the array. The edge of the fin closest to the array was designed to approximate the array shape. The chip further included an on-chip fluid resistor. That is, the inlet and outlet had greater fluid resistance than the array. The height of the device was 139 μm or 142 μm.

Array design: Three-stage symmetrical array gap size:
First stage: 8 μm
Second stage: 12 μm
Third stage: 18 μm
4th stage:
5th stage:
Flow angle: 1/10
Array / chip: 10
Others: Femlab optimized central collection channel (Femlab I), on-chip fluid resistor

  FIG. 54A shows the mask used in making this device. 54B to 54D are enlarged views of the portion of the mask that defines the inlet, the array, and the outlet. 55A to 55S are SEM photographs of actual devices.

Example 10
The device includes one dual array stage having bypass channels arranged to receive effluent from both array ends. The barrier structure of this device is elliptical. The array boundaries were modeled in Femlab to. The chip further included an on-chip fluid resistor. That is, the inlet and outlet had greater fluid resistance than the array. The height of the device was 152 μm.

Array design: One-stage symmetrical array gap size:
First stage: 24 μm
Second stage:
Third stage:
4th stage:
5th stage:
Flow angle: 1/60
Array / chip: 14
Others: Central barrier Elliptical pillar On-chip fluid resistor Femlab modeled array boundary

  FIG. 44A shows the mask used in making this device. 44B to 44D are enlarged views of the portion of the mask that defines the inlet, array, and outlet. 56A to 56C are SEM photographs of actual devices.

Example 11
The deterministic device incorporated in the device of the present invention was designed by computer aided design (CAD) and microfabricated by photolithography. A two-step process has been developed that first reduces the blood sample to remove a large population of small cells and then collects rare target epithelial cells by immunoaffinity capture. The device was defined by photolithography and etched into a silicon substrate based on a design by CAD. A cell enrichment module that is approximately the size of a standard microscope slide has 14 parallel sample processing sections and associated samples connected to a common sample and buffer inlet and product and drain outlets. Has a processing channel. Each section comprises an array of microfabricated barrier structures optimized to separate target cell types by size by moving larger cells to the product stream. In this example, the microchip was designed to separate red blood cells (RBC) and platelets from larger white blood cells and circulating tumor cells. A concentrated population of target cells was recovered from whole blood that passed through the device. The performance of this cell-enriched microchip was evaluated by separating RBCs and platelets from white blood cells (WBC) in normal whole blood (FIG. 67). In cancer patients, circulating tumor cells are found in the larger WBC fraction. Blood was minimally diluted (30%) and 6 mL of sample was processed at a flow rate up to 6 mL / hr. Product stream and Haiekiryu distinguishes automatically different blood cell populations, classified by size, it was assessed by Coulter Model ^"A C -T diff" clinical blood analysis device to count the number. This enrichment chip achieved separation of RBC from WBC, with the WBC fraction retaining more than 99% nucleated cells and removing more than 99% RBC and more than 97% platelets. A representative histogram of these cell fractions is shown in FIG. A normal cytodiagnosis confirmed that the WBC and RBC fractions were highly concentrated (FIG. 69).

  The epithelial cells were then recovered by affinity capture in a microfluidic module functionalized with immobilized antibody. A capture module with a single chamber with a regular array of microfabricated barrier structures coated with antibodies was designed. These barrier structures maximize the capture of cells by expanding the capture region by a factor of about 4 and slowing the flow of cells under laminar flow near the barrier structure to increase the contact time between the cells and the immobilized antibody. It is arranged to become. This capture module can be operated under relatively high flow rate but low shear conditions to prevent cell damage. The surface of the capture module was functionalized by sequential treatment with 10% silane, 0.5% glutaraldehyde and avidin followed by biotinylated anti-EpCAM. The active site was blocked with 3% bovine serum albumin in PBS, quenched with diluted Tris-HCl buffer, and stabilized with diluted L-histidine. The module was washed in PBS after each stage and finally dried and stored at room temperature. The capture performance was measured by human advanced lung cancer cell line NCI-H1650 (ATCC No. CRL-5883). This cell line has an in-frame 15 base pair deletion in heterozygote in exon 19 of EGFR, which makes it gefitinib sensitive. Cells from confluent cultures are harvested with trypsin, stained with the biological dye Cell Tracker Orange (CMRA reagent, Molecular Probes, Eugene, OR), resuspended in fresh whole blood at various flow rates And fractionated with a microfluidic chip. In initial experiments on this feasibility, cell suspensions were processed directly in the capture module without the step of pre-fractionation in the cell concentration module to reduce the red blood cells. Thus, the sample stream contained normal red blood cells and white blood cells in addition to the tumor cells. After processing the cells in the capture module, the device was washed with buffer at a higher flow rate (3 mL / hr) to remove non-specifically bound cells. The adhesive lid was removed, and the adhered cells were fixed with paraformaldehyde on the chip and observed with a fluorescence microscope. The cell recovery rate was calculated from the count with a hemocytometer. Typical capture results are shown in Table 2. The initial yield of reconstitution experiments with unfractionated blood was over 60% and non-specific binding was less than 5%.

Table 2

  Next, NCI-H1650 cells spiked into whole blood and recovered by size fractionation and affinity capture as described above were successfully analyzed in situ. In a pilot experiment to distinguish epithelial cells from leukocytes, 0.5 mL of a stock solution of fluorescently labeled CD45 pan-leukocyte monoclonal antibody was transferred to the capture module and incubated for 30 minutes at room temperature. The module was washed with buffer to remove unbound antibody, and the cells were fixed with 1% paraformaldehyde and observed with a fluorescence microscope. As shown in FIG. 70, the epithelial cells were bound to the barrier structure and bottom of the capture module. Staining of the background of the channel with CD45 pan-leukocyte antibody is seen as with some stained leukocytes, which is apparently due to low levels of non-specific capture.

Example 12 Device Aspect A preferred deterministic device design is shown in FIG. 73A and parameters for three preferred device aspects corresponding to this design are shown in FIG. 73B. These embodiments are particularly useful for separating epithelial cells from blood.

Example 13 PCR analysis of EGFR mutations Blood samples from cancer patients are processed and analyzed using the devices and methods of Example 11 to obtain enriched samples of epithelial cells containing CTCs. This sample is further analyzed to identify potential EGFR mutations.
To perform this analysis, genomic DNA is isolated from target cells present in the enriched sample and amplified for use in allele-specific real-time PCR analysis. EGFR mutations in non-small cell lung cancer that have been reported to date and are known to confer susceptibility or resistance to gefitinib are all within the coding region of exons 18-21. Thus, each of the four exons is PCR amplified with a unique combination of exon specific primers. Allele-specific multiplex quantitative PCR reactions are then performed using the TaqMan 5 ′ nuclease assay PCR system (Applied Biosystems) and the Applied Biosystems real-time PCR device model 7300. This allows any known mutation with clinical significance to be quickly identified.

This method requires a two-step PCR protocol. First, exons 18 to 21 are amplified by a normal PCR reaction. The obtained PCR product is equally divided into samples used for allele-specific multiplex real-time PCR analysis. The initial PCR reaction is stopped during the log phase to minimize the possibility of loss of allele-specific information during amplification. Next, amplification of a partial region specific to each target mutation of the first PCR product is performed by the second stage PCR. The sensitivity of real-time PCR analysis is so high that it is possible to obtain all information about the mutation status of the EGFR gene even if only 10 CTCs have been isolated. Real-time PCR analysis allows quantification of allelic sequences in quantities exceeding 10 ⁸ (8 logs) of input DNA concentration, and thus using this method, heterozygotes in populations with impurities can be quantified. Even mutations are easily detected.

  Oligonucleotides are designed using Primer Express (Applied Biosystems), a primer optimization software, and hybridization conditions are optimized so that the DNA sequence of wild-type EGFR can be distinguished from mutant alleles. Lung cancer cell lines known to have EGFR mutations, H358 (wild type), H1650 (15 base pair deletion, Δ2235-2249), and H1975 (two point mutations, 2369 C → T, 2573 T → G) etc. are used to optimize the allele-specific real-time PCR reaction using EGFR genomic DNA amplified from TaqMan 5 'nuclease assay is used to generate allele-specific labeled probes specific for wild-type sequences or known EGFR mutations. Oligonucleotides are designed to have melting temperatures that can easily distinguish matches from mismatches, and real-time PCR conditions are optimized to distinguish between wild-type and mutant genes. All real-time PCR reactions were performed in triplicate triplicates.

  First, a labeled probe containing a wild type sequence is multiplexed together with one mutant probe in the same reaction. By expressing the result as the ratio of one mutant allele sequence to the wild type sequence, it is possible to distinguish between samples with and without any mutation. By optimizing the conditions for a set of probes, it is then possible to multiplex probes for all mutant alleles in any exon in the same real-time PCR assay, and this analytical tool in the clinical environment Convenience will be improved.

  The purity of the injected CTC sample can vary, and the mutation status of the isolated CTC can also be heterogeneous. However, since real-time PCR is extremely sensitive, any mutant sequences present can be identified.

Example 14 Quantification of the number of different types of cells from epithelial cells By using the method of the present invention, diagnosis based on the number of cell types other than epithelial cells is possible. Diagnosis for the absence, presence, or progression of cancer can be made based on the number of cells in a cell-derived sample that exceed a certain cut-off size. For example, cells with a hydrodynamic size of 14 microns or larger may be selected. This cut-off size will remove most leukocytes. The nature of such cells can then be identified by downstream molecular or cytological analysis.

  Examples of cell types other than epithelial cells that may be useful for analysis include endothelial cells, endothelial progenitor cells, endometrial cells, or trophoblasts that serve as an indicator of disease state. Furthermore, useful diagnostic information can be obtained by separately quantifying the numbers of epithelial cells and other cell types and calculating the ratio between the number of epithelial cells and the number of other cell types.

  As shown in FIGS. 71A-71D, the deterministic device may be configured to isolate a subpopulation of target cells as described above. Most native blood cells, including red blood cells, white blood cells, and platelets, flow into the effluent, and nonnative cells that can contain endothelial cells, endothelial progenitor cells, endometrial cells, or trophoblasts are collected into the concentrated sample. The cut-off size may be selected as follows. This concentrated sample may be further analyzed.

  Thus, by using a deterministic device, it is possible to isolate a subpopulation of cells obtained from blood or other body fluids based on size, so that when targeting large cell types Most of the native blood cells can be easily removed. As schematically shown in FIG. 72, the deterministic device may include a counting means for determining the number of cells in the enriched sample, depending on further analysis of the cells in the enriched sample, diagnostic or other Additional information useful for the purpose can be obtained.

Example 15. Concentration of fetal nucleated red blood cells in maternal blood For example, as described herein, the device comprises a deterministic separator capable of separating fetal nucleated red blood cells and maternal white blood cells from maternal denucleated red blood cells. . This deterministic separation is connected to a reservoir containing sodium nitrite. A maternal blood sample, eg, diluted, is introduced into the device to produce a fraction in which fetal red blood cells are concentrated and maternal red blood cells are removed. This sample is sent to a reservoir where sodium nitrite oxidizes fetal heme iron, thereby increasing the magnetic response of fetal erythrocytes. A magnetic field is then applied, for example by a MACS column, but denatured fetal red blood cells bind to the magnet, whereas maternal white blood cells do not bind to the magnet. By removing the white blood cells, for example by washing, and then removing the magnetic field, fetal red blood cells can be collected for analysis, storage, or further treatment, for example.

Example 16 Separation of Fetal Nucleated Erythrocytes from Blood Using Steep Magnets A typical steep magnet useful for attracting oxyhemoglobin-containing red blood cells is schematically shown in FIG. Red blood cells introduced into the capillaries were collected in separate areas due to the application of a non-uniform magnetic field (FIGS. 79A to 79C).

  FIG. 80 is a photograph of a pellet of nucleated red blood cells (positive fraction) and a pellet of white blood cells (negative fraction) prepared from male umbilical cord blood. Nucleated cells were first extracted from blood using a horizontal deterministic separation device and treated with 50 M sodium nitrite for 10 minutes. The nucleated cells were then passed through a magnetic column to hold nucleated red blood cells. Within the column, the magnetic field strength is about 1 Tesla, the magnetic field gradient is about 3000 Tesla / m, and the flow rate is about 0.4 mm / sec. Met. Leukocytes were washed from the column with Dulbecco's PBS buffer containing 1% BSA and 2 mM EDTA and collected as a negative fraction. Nucleated erythrocytes were eluted from the column using the same buffer at a flow rate of 4 mm / s and collected as a positive fraction.

  FIG. 81 is a series of fluorescent images of nucleated red blood cells isolated from maternal blood by the method shown in FIG. Cells were stained by fluorescence in situ hybridization (FISH). The X chromosome was identified with an indigo green labeled probe for the alpha satellite region, while the Y chromosome was identified by red and green staining for the alpha satellite region and satellite III region, respectively. Nuclei were counterstained with DAPI (blue).

  FIG. 82 shows various mature stages of nucleated red blood cells isolated from maternal blood using the method shown in FIG. Cells were stained by light Giemsa staining.

  83A and 83B are (A) a photomicrograph showing the result of concentration using anti-CD71 antibody, and (B) a photomicrograph showing the result of concentration using the method shown in FIG. The sample of (A) contained more than 200,000 nucleated cells per mL of blood, and the sample of (B) contained about 100 to 500 nucleated cells per mL of blood. The purity of nucleated red blood cells obtained by the method shown in FIG. 80 was about 1000 times better than the concentration method using an antibody.

  FIG. 101 shows a photomicrograph of fetal cells with chromosome 21 trisomy obtained by deterministic device and continuous enrichment by magnetic enrichment.

Example 17. Exemplary Methods for Cell Concentration Three methods for practicing the preferred embodiments of the present invention are shown in FIG. For affinity enrichment, the sample is passed through a deterministic device as described herein. The deterministic device effluent is then contacted with magnetic beads coated with antibodies or other selective binding components. Cells associated with the beads are then subjected to magnetic separation, cytospin, and analysis, eg, by FISH. For hemoglobin concentration, the sample is passed through a deterministic device as described herein. The effluent of the deterministic device is then contacted with a reagent capable of oxidizing hemoglobin, such as sodium nitrite, to magnetically isolate the magnetically responsive cells. Separated cells may be subjected to cytospin and analysis, eg, by FISH, or may be subjected to molecular analysis. In the integrated method, the device of the present invention comprises a deterministic enrichment and a magnetic enrichment from which the effluent may be subjected to molecular analysis. FIG. 85 schematically shows an integrated device of the present invention.

Example 18 Selective lysis after enrichment The following examples focus on the extraction of nuclear populations of circulating fetal cells obtained from maternal whole blood, but the described method is based on the extraction of cellular components from other cells. Also widely used for isolation.

Isolation of Fetal Cell Nuclei FIG. 86 is a flowchart showing a method for isolating fetal cell nuclei from a maternal blood sample. Red blood cells can be selectively lysed by this method (FIG. 87).

  Several embodiments of a method for isolating a purified nuclear population of circulating cells subject to genome analysis from whole blood are shown below.

(A) The method includes microfluidic processing of whole blood, as described herein. This treatment consists of (1) preparing a sample enriched with nucleated cells by removing 10 ¹ -10 ³ (1 to 3 log) times the number of enucleated red blood cells and platelets, and (2) this enriched nucleated sample. In addition, fetal cell nuclei are released by microfluidic treatment for selectively lysing residual enucleated erythrocytes, denucleated erythrocytes, and nucleated erythrocytes with respect to maternal nucleated leukocytes. To separate nuclei from maternal nucleated leukocytes by microfluidic processing, and (4) to analyze the fetal genome using commercially available genetic analysis tools.

  (B) The method may be designed so that steps 1 and 2 of aspect 1 are taken as a step with a microfluidic device, followed by a step 3 using a component of a downstream device or a larger device. Good (see FIGS. 88 and 89). FIG. 88 shows a schematic diagram of a microfluidic device for simultaneously performing size concentration and dissolution. This device has two barrier structure regions that deflect larger cells from the end of the device into which the sample is introduced to a central channel containing lysate (eg, the dual device described herein). In the case of maternal blood, the area of the barrier structure is positioned so that maternal enucleated red blood cells and platelets remain at the end of the device, while fetal nucleated red blood cells and other nucleated cells are deflected to the central channel. When deflected to the central channel, fetal red blood cells (target cells) are lysed. FIG. 89 is a diagram schematically showing a microfluidic device for separating nuclei (target cell-derived components) from undissolved cells based on size. This device is similar to the device shown in FIG. 88 except that a barrier structure is placed to hold the nucleus at the device edge while deflecting large particles into the central channel.

  (C) Bulk processing methods such as density gradient centrifugation to separate fetal cell nuclei from maternal cells following a combination method for fetal cell nuclei generation in a maternal blood sample based on microfluidics (see FIG. 90).

(D) Method and principle verification
Selective Lysis and Fractionation of Nucleated Red Blood Cells Contaminated red blood cells in donor blood samples spiked with normal umbilical cord blood were lysed in two ways: hypotonic lysis and ammonium chloride lysis. Since enucleated erythrocytes lyse faster than nucleated cells in hypotonic solutions, the time of exposure of the mixed cell population in hypotonic solutions can be controlled to select the cell population based on this time. Dissolution is possible. In this method, cells are pelleted and the plasma on the pellet is aspirated. Deionized water is then added to mix the pellet with water. An exposure of 15 seconds is sufficient to lyse more than 95% of enucleated erythrocytes with minimal nucleated red blood cell lysis, and 15 to 30 seconds is sufficient to lyse more than 70% of nucleated red blood cells. However, the lysis of other nucleated cells is less than 15%, and the lysis rate of other nucleated cells increases after 30 seconds. After the desired exposure time, 10 × HBSS (hyperosmotic balanced salt) solution is added to return the solution to an isotonic state. Exposure to a standard concentration (eg, 0.15M isotonic solution) ammonium chloride lysis solution can lyse most of the red blood cells with minimal effect on nucleated cells. If the osmolarity of this lysis solution is lowered to make a low osmotic ammonium chloride solution, most nucleated red blood cells are lysed together with mature red blood cells.

  Enriched lymphocyte populations were obtained using density centrifugation. An aliquot of these lymphocyte samples was exposed to hypotonic ammonium chloride solution for a time sufficient for more than 95% of cells to lyse. These nuclei were then labeled with Hoechst 33342 (bisbenzimide H33342), a dye specific for the AT-rich region of double-stranded DNA, and returned to the original lymphocyte population in 90:10 (cell: nucleus). A mixture was made. This mixture was introduced into a device that separates cells from nuclei based on size as shown in FIG. 89, the drainage and product fractions were collected, and the cell: nuclear ratio contained in each fraction was measured. .

The lysis nuclei of the mixed cell suspension treated in the density gradient centrifugation selective lysis step of the lysed product can be concentrated by adding a sucrose cushion solution to the lysate. This mixture is then layered onto pure sucrose cushion solution and then centrifuged to form a concentrated nuclear pellet. Undissolved cells and debris are aspirated from the supernatant. Resuspend nuclear pellet in buffer and cytospin onto glass slide.

Nuclear RNA-FISH method for acid alcohol whole cell lysis and target cell identification The product obtained from a device that separates cells based on size as shown in FIG. Methanol: acetic acid = 3: 1) was exposed for 30 minutes to lyse more than 99% enucleated cells and more than 99.0% nucleated cells. Prior to acid alcohol lysis, a low osmotic treatment may be performed in which the cells are exposed to a salt solution (0.6% NaCl) for 30 minutes to swell the nucleus. The released nuclei can be quantitatively deposited on the glass slide by cytospin and FISH can be performed (FIGS. 95a and 95b). Target cells such as fetal nucleated erythrocytes are analyzed by RNA-FISH method using positive selection probes such as zeta, epsilon and gamma-globin and negative selection probes such as beta-globin, or the length of telomeres is analyzed. Can be identified. Other methods of distinguishing between fetal and non-fetal cells are known in the art, for example as described in US Pat. No. 5,766,843.

Example 19. Single Stage Size Separation Device FIG. 91 shows a size separation device optimized to separate particles in blood. This is a single stage device with a fixed width gap of 22 μm, with 48 multiplexed arrays for simultaneous sample processing. The device parameters are as follows.

  Blood was collected from pregnant volunteer volunteers and diluted 1: 1 with Dulbecco's phosphate buffer (without calcium and magnesium) (iDPBS). Blood and flow buffer (iDPBS with 1% BSA and 2 mM EDTA) were fed into the device connected to the manifold shown in Example 20 at 0.8 PSI active pressure. The blood was separated into two components: nucleated cells in the flow buffer and enucleated cells and plasma proteins in the flow buffer. Both components were analyzed with a standard impedance meter. Components comprising nucleated cells were further characterized using propidium iodide staining solution in combination with a standard Nagiotte counting chamber to determine total nucleated cell loss. Using the obtained data, blood processing volume (mL), blood processing rate (mL / hr), RBC and platelet removal rate, and nucleated cell retention rate were calculated. The following table shows the results of cell enrichment using this device.

Example 20. Manifold used for size separation device A typical manifold into which the microfluidic device of the present invention is inserted is shown in FIG. The manifold is divided in half and consists of two parts, between which the microfluidic device of the present invention is placed. One half of the manifold has separate inlets for blood and buffer, each connected to a corresponding fluid reservoir. The channel in the device is arranged to be connected to the reservoir through the through hole of the device. Generally, the device is placed vertically and the treated blood is collected as it drops from the product stream outlet. Furthermore, in order to limit the size of the droplet formed in the region around the product flow outlet of the microfluidic device, it may be marked with a hydrophobic material, for example with a permanent marker. The device further comprises two hydrophobic vent filters, for example a 0.2 μm PTFE filter. This filter displaces bubbles that have entered the device with an aqueous solution, but does not allow liquids to pass under low pressure, eg, below 5 psi.

  For pre-operation of the device, Dulbecco's PBS buffer with buffer, eg 1% (weight / volume) bovine serum albumin and 2 mM EDTA, is degassed for 5-10 minutes with stirring under reduced pressure. The buffer is then pumped from the manifold buffer inlet into the device at a pressure of less than 5 psi. Subsequently, the buffer is filled into the buffer chamber by replacing air through a hydrophobic vent filter, and further fills the channels and blood chambers in the microfluidic device. Indoor air can be excluded by a hydrophobic vent filter connected to the blood chamber. After filling the blood chamber, the buffer is sent to the blood inlet. In some embodiments, after a 1 minute pre-run with a pressure of 1 psi, the blood inlet is closed and the pressure is increased to 3 psi for 3 minutes.

Example 21. Hypoosmotic lysis after concentration A fetal nRBC population concentrated with any of the devices described herein is subjected to hypotonic shock by adding a large volume of low ionic strength buffer, such as deionized water, And selectively dissolve nRBCs to release their nuclei. Subsequently, the hypotonic shock is stopped by adding the same volume of high ionic strength buffer. The released nuclei may be subsequently recovered by gradient centrifugation, such as by passing an aqueous solution of iodixanol of ρ = 1.32 g / mL. Analyze the released nuclei.

  FIG. 93 shows the selective lysis of fetal nRBC relative to maternal nRBC as a function of exposure time to lysis conditions. This selective lysis procedure can also be used to selectively lyse fetal nRBC in a cell population consisting of fetal nRBC, maternal nRBC, enucleated fetus and maternal nRBC, and fetal and maternal white blood cells. Fetal nRBC and maternal nRBC were dissolved by applying hypotonic shock for a certain period of time using distilled water, and then stopping the shock by adding the same volume of a 10-fold salt solution such as PBS. Meanwhile, the number of lysed (non-viable) fetal nRBCs increased 10-fold, while the number of lysed maternal nRBCs increased by a smaller factor. At any time point, lysed cells were stained with propidium iodide and concentrated by gradient centrifugation to determine the ratio of lysed fetal nRBC to maternal nRBC. An optimal time for selective enrichment of fetal nRBC nuclei can be determined and applied.

Example 22. Selective lysis of maternal cells Fetal nRBC populations enriched with any of the devices described herein can be subjected to selective lysis of maternal red blood cells.

To selectively lyse enucleated RBCs and maternally nucleated RBCs, samples enriched with fetal nRBC were prepared using 0.155 M NH ₄ Cl, 0.01 M KHCO ₃ , 2 mM EDTA, acetazolamide (eg, 0. Treatment with an RBC lysis buffer such as 1% BSA with a carbonic anhydrase inhibitor (such as 1 mM to 100 mM) followed by 10 volumes of 1 × PBS or 4,4′-diisothiocyanostilbene-2 Stop the lysis step with a large volume of balanced salt buffer, such as 1 × PBS with an ion exchange inhibitor such as 2′-disulfonic acid (DIDS). Viable fetal cells may then be subjected to further sorting and analysis.

K562 cells for mimicking leukocytes were labeled with Hoechst and calcein AM for 30 minutes at room temperature (FIG. 94). The labeled K562 cells were added to the blood sample and further buffer (0.155 M NH ₄ Cl, 0.01 M KHCO ₃ , 2 mM EDTA, 1% BSA, and 10 mM acetazolamide) was added (spike with buffer). The volume ratio with the blood was 3: 2). Spiked blood samples were incubated for 4 hours at room temperature with periodic gentle agitation. The fraction of living cells in each spiked specimen was quantified by measuring green fluorescence at 610 nm at multiple time points. Cell lysis is observed only 3 minutes after treatment (in the absence of DIDS).

Example 23. Genetic analysis For example, by any device or method discussed herein, a sample enriched in fetal nRBC can be lysed and analyzed for genetic content. Examples of methods that can be used for cell lysis and isolation of desired cells or cell components include the following.
(A) A sample enriched with fetal nRBC may be subjected to whole cell lysis to remove cytoplasm and isolate nuclei. The nuclei may be immobilized by treating with a fixative such as Carnoy's fixative and adhering to a slide glass. The fetal nucleus can be identified by the presence of a target endogenous to the fetus by immunostaining of nuclear proteins and transcription factors, or by differential hybridization of fetal mRNA precursors or RNA FISH (Gribnau et al. Mol. Cell, p. 377-386 (2000), Osborne et al., Nat. Gene., P. 1065-1071 (2004), Wang et al., Proc. Natl. Acad. Sci., P. 7391-7395 (1991). ), Alfonso-Pizzarro et al., Nucleic Acids Research, p. 8363-8380 (1984)). The fetal endogenous targets include zeta-, epsilon-, gamma-, delta-, beta-, alpha-globin, and I-branching enzyme (Yu et al., Blood, Vol. 101, p. .2081, (2003)), N-acetylglucosamine transferase, or non-globin targets such as IgnT. Oligonucleotide probes used in the RNA FISH method may be against intron-exon boundaries or other regions, or the necessary targets are uniquely identified by analyzing telomere length.
(B) A sample enriched with fetal nRBC may be selectively lysed using treatment with a buffer and an ion exchange inhibitor as described in Example 22 to isolate fetal cells. Viable fetal cells may be further screened by the presence or absence of intracellular markers such as globin and I-branched beta 1,6-N-acetylglucosaminyltransferase or surface markers such as antigen I. In another embodiment, the concentrated fetal nRBC is selectively lysed as described in Example 22 to remove both the enucleated RBC and maternal nRBC, followed by antibodies against the surface antigen CD45 present in all nucleated leukocytes. The complement-mediated cell lysis used may be performed. The resulting intact fetal nRBC should not contain any other contaminating cells.
(C) The fetal nRBC enriched sample may be lysed by hypotonic shock as described in Example 21 to isolate fetal cell nuclei. The nucleus may be fixed by treating with a fixative such as Carnoy fixative and adhering it to the slide glass.

  After isolation, the desired cells or cell components (eg nuclei) may be analyzed for genetic content. The FISH method may be used to identify defects in chromosomes 13 and 18 or other chromosomal abnormalities such as chromosome 21 trisomy or XXY. Chromosome aneuploidy can also be detected by methods such as comparative genomic hybridization. In addition, the identified fetal cells may be examined by microdissection. After extraction, fetal cell nucleic acid is subjected to one or more PCRs or whole genome amplification, followed by comparative genomic hybridization or short tandem repeat (STR) analysis, or single base point mutation (SNP), deletion Alternatively, gene mutation analysis such as translocation may be performed.

Example 24. Lysis after size concentration A product comprising 3 mL of red blood cells in 1 × PBS, obtained from the device shown in FIG. 89, is treated with 50 mM sodium nitrite / 0.1 mM acetazolamide for 10 minutes. The cells are then contacted with a lysis buffer consisting of 0.155 M NH ₄ Cl, 0.01 M KHCO ₃ , 2 mM EDTA, 1% BSA, and 0.1 mM acetazolamide, and 4,4′-diiso The dissolution reaction is stopped by adding dropwise directly to a reaction terminator containing a band 3 ion exchanger channel inhibitor such as thiocyanostilbene-2,2′-disulfonic acid (DIDS). Enucleated RBCs and nucleated RBCs are counted after light Giemsa staining, and the FISH method is used for nRBC counting. The resulting numerical value is then compared to an undissolved control sample. One of the results of such an experiment is shown below.

Example 25. Concentration of apoptotic DNA Chaotropic salt or detergent-mediated total lysis and oligonucleotide-mediated enrichment of apoptotic DNA from fetal nucleated RBCs The product obtained from the device shown in FIG. 89 was prepared with a buffered guanidinium hydrochloride solution (at least 4. 0M), guanidinium thiocyanate (at least 4.0M), or in a chaotropic salt solution such as a buffered surfactant solution such as SDS-added Tris buffer solution. The cell lysate is then incubated with 10 μl of 50 mg / ml protease K at 55 ° C. for 20 minutes to remove protein, followed by incubation at 95 ° C. for 5 minutes to inactivate the protease. Fetal nRBC undergoes apoptosis when it enters the maternal blood circulation, and this process of apoptosis causes DNA fragmentation of fetal nRBC DNA. Utilizing the high efficiency of separating smaller DNA fragments from intact genomic DNA by reducing the size of fetal nRBC DNA and oligonucleotide-mediated enrichment, apoptotic fetal nRBC DNA was attached to beads, in solution, or in arrays or others Can be selectively enriched by hybridization to identify unique molecular markers, such as short tandem repeats (STR), to oligonucleotides bound to the surface. After hybridization, unwanted nucleic acids or other impurities are washed away with a high salt buffer, such as a solution comprising 150 mM sodium chloride in 10 mM Tris-HCl, pH 7.5, after which the captured target is washed with 10 mM at pH 7.8. Release into buffer such as Tris buffer or distilled water. The apoptotic DNA thus enriched is then analyzed using genetic content analysis methods such as those described in Example 23, for example.

Example 26. Lysis Procedure FIG. 96 is a flowchart showing details of variations of the lysis step that can be used for the maternal blood sample. Although illustrated as starting from a concentrated product, such as a product made with the devices and methods described herein, this step can be performed on any maternal blood sample. The lysis step consists of (i) selecting required cells (eg fetal cells), (ii) selecting required cells and their nuclei, (iii) all cells, (iv) all cells and (V) unwanted cells (eg, maternal RBC, WBC, platelets, or combinations thereof), (vi) unwanted cells and their nuclei, and (vii) nuclei of all cells and unwanted cells. The chart shows that can be used to dissolve selectively. The flowchart further shows an exemplary method for isolating the released nuclei (the devices and methods of the present invention may also be used for this purpose) and an exemplary method for analyzing the results.

Example 27. Total cell lysis titration
This is an example of titration of whole cell lysis in a microfluidic environment. A blood sample concentrated by the size separation method described herein was divided into four samples of the same volume. Three samples were processed in a microfluidic device capable of transporting cells into a defined first medium of defined flow path length within the device and subsequently into a defined second collection medium. . The volume flow rate of the cell suspension was varied to control the incubation time within a defined flow path length in the predefined first medium before contacting the predefined second medium. In this example, deionized water was used as the default first medium and 2 × PBS was used as the default second medium. The flow rate was adjusted so that the incubation time in deionized water was 10 seconds, 20 seconds, or 30 seconds before the cells were mixed with 2 × PBS to produce an isotonic solution. Total cell counts in three treated samples and one untreated sample were counted with a hemocytometer.

Example 28. Magnetic Concentration Device This example provides a typical device for isolating magnetic cells with low non-specific carryover using a magnetic column. Target cells are labeled with magnetic beads or converted to paramagnetism using a chemical reagent such as NaNO ₂ .

  A diagram of the device is shown in FIG. 97, and a photograph is shown in FIG. The device comprises a magnetic column, a fluid reservoir connected to one end of the column, a valve that controls the fluid connection at the other end of the column, a flow rate control device, a buffer vessel, and a drain vessel.

  The device may be used as shown below.

1. The cell sample is put into the reservoir.
2. Connect the magnetic column to the flow rate control device using the valve appropriately. Apply a magnetic field to the column. The cell sample in the reservoir is passed through the column at a controlled low flow rate by the flow rate control device. Magnetically responsive cells are retained in the column.
3. Optionally, buffer is supplied to the reservoir to wash the column (under magnetic field).
4). Change the valve settings so that the magnetic column is fluidly connected to the buffer vessel.
5. Optionally, the application of the magnetic field to the column is stopped.
6). Wash the column by backflowing the buffer at a high flow rate from the buffer container to the reservoir. Nonspecifically remaining cells in the column are washed away to the reservoir.
7). Repeat steps 2 to 6 as necessary.
8). The target cells are eluted at a high flow rate toward the reservoir.
9. Alternatively, the target cell is eluted by removing the magnetic column from the magnetic field and washing the column with a buffer introduced through the reservoir.

Example 29. Magnetic enrichment of nucleated red blood cells The following examples show data relating to the enrichment of nRBC from maternal blood.

Sample preparation:
20 mL each of blood from 8 separate consent providers is collected into K ₂ EDTA anticoagulant tubes and processed within 6 hours with the size separation device described herein, and nucleated red blood cells in buffer (iDPBS, 1% Of BSA, 2 mM EDTA).

Magnetic concentration step:
1. The concentrated cell sample is put into the reservoir.
2. As a valve setting, connect the magnetic column to a flow rate control device. A 1.4 Tesla magnetic field was applied to the column. The cell sample in the reservoir is passed through the column at about 0.3 mm / s by the flow rate control device.
3. Supply 3 mL of buffer to the reservoir to wash the column (under magnetic field).
4). Change the valve settings so that the magnetic column is fluidly connected to the buffer vessel.
5. The column is washed by backflowing the buffer from the buffer container toward the reservoir at about 60 mm / s.
6). Repeat steps 2 to 5 twice.
7. The target cells are returned to the reservoir and eluted at 60 mm / s using 3 mL of buffer.
8). Alternatively, the target cell is eluted by removing the magnetic column from the magnetic field and rinsing the column with buffer introduced through the reservoir.

result:
Purified cells were counted for nRBC and WBC. Overall, more than 99.95% WBC is removed by this process, and nRBCs are collected with a frequency exceeding 2 nRBCs per mL of whole blood in all cases.

Example 30. FIG. Yield of Magnetic Concentration In this example, a mixture of white blood cells (WBC) and red blood cells (RBC) is used as a sample, and the yield is shown when the hemoglobin-containing cells are magnetically separated. RBC is a target cell to be enriched, and WBC is a cell to be removed.

Sample preparation:
20 mL of blood from the consent donor is collected into a K ₂ EDTA anticoagulant tube and processed within 6 hours with the size separation device described herein to buffer nucleated red blood cells and some RBC carryover (iDPBS, 1% In BSA, 2 mM EDTA). Samples were measured for WBC count (16.2 million) and RBC count (7.6 million).

Magnetic concentration step:
1. The cell sample is put into the reservoir.
2. As a valve setting, connect the magnetic column to a flow rate control device. A 1.4 Tesla magnetic field was applied to the column. The cell sample in the reservoir is passed through the column at a flow rate of about 0.3 mm / s by the flow rate control device.
3. Supply 3 mL of buffer to the reservoir to wash the column (under magnetic field).
4). Change the valve settings so that the magnetic column is fluidly connected to the buffer vessel.
5. The column is washed by backflowing the buffer from the buffer container toward the reservoir at about 60 mm / s.
6). Repeat steps 2 to 5 twice.
7. The target cells are returned to the reservoir and eluted at 60 mm / s using 3 mL of buffer.

result:
Purified cells were counted for RBC and WBC. The number of RBCs was 7.5 million and the number of WBCs was 3200. Therefore, the yield of target cells (RBC) was over 98%, and 99.98% non-target cells (WBC) were removed.

Example 31. Selective Lysis This example provides a method for purifying nucleated red blood cells with low RBC carryover by selective lysis of RBCs. Selective lysis can occur before, during, or after magnetic separation.

Step (selective dissolution before magnetic separation):
1. Starting with whole blood or enriched nucleated cells, nucleated red blood cells are selectively lysed using a chemical reagent such as NH ₄ Cl.
2. Treat nucleated cells in NaNO ₂ (about 50 mM) for 5 minutes.
3. Nucleated red blood cells are separated from other nucleated cells using the magnetic separation device described herein.

Step (selective dissolution after magnetic separation):
1. Starting with whole blood or concentrated nucleated cells, the nucleated cells are treated in NaNO ₂ (about 50 mM) for 5 minutes.
2. Nucleated red blood cells are separated from other nucleated cells using the magnetic separation device described herein.
3. Enucleated erythrocytes are selectively lysed using a chemical reagent such as NH ₄ Cl.

Step (selective dissolution during magnetic separation):
1. Starting with whole blood or concentrated nucleated cells, the nucleated cells are treated in NaNO ₂ (about 50 mM) for 5 minutes.
2. Nucleated erythrocytes are separated from other nucleated cells using a magnetic separation device (magnetic column holding nRBC). A chemical reagent such as NH ₄ Cl is flowed through the column to selectively lyse the enucleated erythrocytes. An inert reagent is flowed through the column to stop the lysis reaction.
3. nRBC is eluted from the column.

  Due to the dissolution during the separation, the time of the dissolution reaction can be precisely adjusted.

Example 32. Lysis for nuclei preparation This example provides a method for isolating nucleated red blood cell nuclei from blood using magnetic enrichment and whole blood lysis. The method includes providing a concentrated nucleated red blood cell sample obtained by magnetic separation, and the sample is acid alcohol (methanol and acetic acid 3: 1) or Triton solution (0.2% Triton in iDPBS). Or the like) may be brought into contact with a chemical reagent such as

Other Embodiments All publications, patents, and patent applications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific embodiments, it is to be understood that the invention is not unduly limited to such specific embodiments, as claimed. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be included in the scope of the present invention.

  Other aspects are in the claims.

1A to 1E are diagrams schematically showing an array that separates cells based on deterministic movement in the horizontal direction, and (A) shows a shift in the horizontal direction of each adjacent row, (B) (C) Analytes having a hydraulic size above the critical size are moved laterally in the apparatus, representing the fluid flowing through the gaps non-uniformly diverting around the barrier structures in adjacent rows It is a figure showing the array by the (D) elliptical barrier structure showing the state, (D) showing the array by the cylindrical barrier structure. 1A to 1E are diagrams schematically showing an array that separates cells based on deterministic movement in the horizontal direction, and (A) shows a shift in the horizontal direction of each adjacent row, (B) (C) Analytes having a hydraulic size above the critical size are moved laterally in the apparatus, representing the fluid flowing through the gaps non-uniformly diverting around the barrier structures in adjacent rows It is a figure showing the array by the (D) elliptical barrier structure showing the state, (D) showing the array by the cylindrical barrier structure. 1A to 1E are diagrams schematically showing an array that separates cells based on deterministic movement in the horizontal direction, and (A) shows a shift in the horizontal direction of each adjacent row, (B) (C) Analytes having a hydraulic size above the critical size are moved laterally in the apparatus, representing the fluid flowing through the gaps non-uniformly diverting around the barrier structures in adjacent rows It is a figure showing the array by the (D) elliptical barrier structure showing the state, (D) showing the array by the cylindrical barrier structure. FIG. 2 is a diagram schematically illustrating a state in which the fluid flowing through the gaps is unequally divided around the barrier structures in adjacent rows. FIG. 3 is a diagram schematically showing how the critical size is affected by the flow profile (in this example, parabolic). FIG. 4 illustrates how the shape affects the movement of the analyte within the device. FIG. 5 illustrates how deformability affects the movement of analytes within the device. FIG. 6 is a diagram schematically illustrating the deterministic movement in the lateral direction. Analytes with a hydrodynamic size greater than the critical size move to the end of the array, while analytes with a hydrodynamic size less than the critical size pass through the device without lateral movement. FIG. 7 is a diagram schematically showing a three-stage deterministic device. FIG. 8 is a diagram schematically illustrating the maximum size and the cutoff size in the device of FIG. FIG. 9 is a diagram schematically illustrating a bypass channel. FIG. 10 is a diagram schematically illustrating a bypass channel. FIG. 11 is a schematic representation of a three-stage deterministic device having a common bypass channel. FIG. 12 is a schematic representation of a three-stage double deterministic device with a common bypass channel. FIG. 13 is a schematic representation of a three-stage deterministic device with a common bypass channel and a substantially constant flow rate within the device. FIG. 14 is a schematic representation of a three-stage dual deterministic device with a common bypass channel and a substantially constant flow rate within the device. FIG. 15 is a schematic representation of a three-stage deterministic device having a common bypass channel and having substantially constant fluid resistance in the bypass channel and in adjacent stages. FIG. 16 is a schematic representation of a three-stage double deterministic device having a common bypass channel and having substantially constant fluid resistance in the bypass channel and in adjacent stages. FIG. 17 is a schematic representation of a three-stage deterministic device with two independent bypass channels. FIG. 18 is a schematic representation of a three-stage deterministic device having two independent bypass channels having an arbitrary shape. FIG. 19 is a schematic representation of a three-stage dual deterministic device with three independent bypass channels. FIG. 20 is a schematic representation of a three-stage deterministic device having two independent bypass channels and the flow rate of each stage being substantially constant. FIG. 21 is a schematic representation of a three-stage double deterministic device with three independent bypass channels and the flow rate of each stage is substantially constant. FIG. 22 is a diagram schematically illustrating the fluid extraction interface. FIG. 23 is a diagram schematically illustrating the fluid supply interface. FIG. 24 is a diagram schematically illustrating the flow supply interface including the bypass channel. FIG. 25 is a diagram schematically illustrating two flow supply interfaces adjacent to the central bypass channel. FIG. 26 is a schematic representation of a device having four channels that act as on-chip fluid resistors. FIG. 27 is a diagram schematically illustrating the influence of the on-chip resistor on the relative width of two fluids flowing in the device. FIG. 28 is a diagram schematically illustrating the influence of the on-chip resistor on the relative width of two fluids flowing in the device. FIG. 29 is a diagram schematically illustrating a dual device having one common inlet for two external regions. FIG. 30A schematically illustrates a duplex array on a device. FIG. 30B schematically illustrates a plurality of arrays having a common inlet and product stream outlet on the device. FIG. 31 is a diagram schematically illustrating a multistage device having a small occupation area. FIG. 32 is a diagram schematically showing blood flowing in the device. FIG. 33 is a graph showing the hydrodynamic size distribution of blood cells. FIGS. 34A-34D show analyte transfer from sample to buffer in a single stage (A), three stage (B), dual (C), three stage dual (D) deterministic device. It is a figure showing typically. FIGS. 34A-34D show analyte transfer from sample to buffer in a single stage (A), three stage (B), dual (C), three stage dual (D) deterministic device. It is a figure showing typically. FIGS. 34A-34D show analyte transfer from sample to buffer in a single stage (A), three stage (B), dual (C), three stage dual (D) deterministic device. It is a figure showing typically. FIGS. 34A-34D show analyte transfer from sample to buffer in a single stage (A), three stage (B), dual (C), three stage dual (D) deterministic device. It is a figure showing typically. FIG. 35A is a schematic representation of a two-stage deterministic device used to move particles from blood into a buffer to obtain three products. FIG. 35B is a graph schematically showing the maximum size and the cutoff size of the two stages. FIG. 35C is a graph schematically showing the composition of the three products. FIG. 36 is a schematic representation of a two-stage deterministic device for denaturation where each stage has a bypass channel. FIG. 37 schematically illustrates the use of a fluid channel to connect two stages in the device. FIG. 38 is a diagram schematically illustrating the use of a fluid channel to connect two stages in the device when the two stages are formed as an array having a small occupation area. FIG. 39A schematically illustrates a two-stage deterministic device having a bypass channel that receives effluent from both stages. FIG. 39B is a graph that schematically represents the product size range achievable with the device. FIG. 40 schematically illustrates a two-stage deterministic device for denaturation having a bypass channel adjacent to each stage and exiting to the same outlet. FIG. 41 is a schematic representation of a deterministic device for the sequential movement and denaturation of particles. FIG. 42A is a photograph of a deterministic device that may be incorporated into the device of the present invention. 42B-42E are masks used in the manufacture of devices that may be incorporated into the present invention. FIG. 42F is a set of photographs of the device containing blood and buffer. 43A-F are obtained by the blood analysis device for the blood sample and the drainage fraction (buffer, plasma, red blood cells and platelets) and product fraction (buffer and nucleated cells) obtained by the device of FIG. This is a typical histogram. 44A-44D are masks used in the manufacture of deterministic devices that may be incorporated into the devices of the present invention. 45A-45D are masks used in the manufacture of deterministic devices that may be incorporated into the devices of the present invention. FIG. 46A is a photomicrograph of a sample enriched in fetal erythrocytes. FIG. 46B is a photomicrograph of maternal red blood cell drainage. FIG. 47 is a series of photomicrographs showing positive selection of male fetal cells (blue represents the nucleus, red represents the X chromosome, and green represents the Y chromosome). FIG. 48 is a series of photomicrographs showing gender and positive selection of chromosome 21 trisomy. 49A-49D are masks used in the manufacture of deterministic devices that may be incorporated into the devices of the present invention. 50A-50G are electron micrographs of the device of FIG. 50A-50G are electron micrographs of the device of FIG. 50A-50G are electron micrographs of the device of FIG. 50A-50G are electron micrographs of the device of FIG. 51A-51D are masks used in the manufacture of deterministic devices that may be incorporated into the devices of the present invention. 52A-52F are electron micrographs of the device of FIG. 52A-52F are electron micrographs of the device of FIG. 52A-52F are electron micrographs of the device of FIG. 53A-53F are electron micrographs of the device of FIG. 53A-53F are electron micrographs of the device of FIG. 53A-53F are electron micrographs of the device of FIG. 53A-53F are electron micrographs of the device of FIG. 54A-54D are masks used in the manufacture of deterministic devices that may be incorporated into the devices of the present invention. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 55A-55S are electron micrographs of the device of FIG. 56A-56C are electron micrographs of the device of FIG. 56A-56C are electron micrographs of the device of FIG. FIG. 57A is a schematic diagram illustrating a deterministic device that may be incorporated into the device of the present invention and its operation. FIG. 57B is a diagram of the device of FIG. 57A and a schematic representation of this device. Figures 58A and 58B show two different arrangements for connecting two deterministic devices to each other. FIG. 58A shows a cascade type in which the outlet 1 of one device is connected to the sample inlet of a second device. Figures 58A and 58B show two different arrangements for connecting two deterministic devices to each other. FIG. 58B shows a bandpass type where the outlet 2 of one device is connected to the sample inlet of the second device. FIG. 59 is a diagram showing an improved size separation method in which target cells are labeled with immunoaffinity beads. FIG. 60 is a diagram showing a method of performing size fractionation using a device that may be incorporated in the present invention, and a method of separating a free labeling reagent such as an antibody from a bound labeling reagent. FIG. 61 is a diagram showing the method of FIG. In this case, the non-target cell may be co-purified with the target cell, but the non-target cell does not interfere with the quantification of the target cell. FIG. 62 is a diagram showing a method of preparing a concentrated sample of these cells by separating large cells from the mixture. FIG. 63 is a diagram showing a method of lysing cells in the device of the present invention to separate whole cells from organelles and other cell-derived components. FIG. 64 is a diagram showing two devices that are arranged in cascade and used for size fractionation and separation of free labeled reagent from bound labeled reagent using the device of the present invention. FIG. 65 is a diagram showing two devices that are arranged in cascade and used for size fractionation and separation of free labeled reagent from bound labeled reagent using the device of the present invention. In this figure, phage rather than antibody is used for binding and detection. FIG. 66 is a diagram showing two devices arranged in a bandpass type. FIG. 67 is a graph of cell number against hydrodynamic cell diameter in microfluidic separation of normal whole blood. Figure 68 is a clinical blood analysis device "Coulter A ^C -T diff" in the resulting infusion samples, generating a sample, and a histogram set of drainage samples. The x-axis is the amount of cells expressed in femtomole. FIG. 69 is a set of representative photomicrographs of fetal blood product stream and drainage stream treated with a cell enrichment module showing distinct separation of nucleated cells and red blood cells. FIG. 70 is an image of cells fixed with paraformaldehyde on a cell concentration module and observed with a fluorescence microscope. Target cells are coupled to the barrier structure and floor of the capture module. FIG. 71A is a graph of cell number versus hydrodynamic cell diameter in microfluidic separation of normal whole blood. FIG. 71B is a graph of cell number versus hydrodynamic cell diameter in microfluidic separation of whole blood with circulating tumor cell populations. FIG. 71C is a graph further showing a cutoff size that excludes most natural blood cells from the graph of FIG. 71B. FIG. 71D further illustrates that the graph of FIG. 71C may include endothelial cells, endometrial cells, or trophoblasts that indicate a disease state in a cell population that is larger than the cutoff size. It is a graph. FIG. 72 is a schematic diagram of a method for isolation and enumeration of large cells in a cell-derived sample, and subsequent further analysis of large cell subpopulations. Here, the number of large cells indicates the disease state of the patient. FIG. 73A illustrates a suitable deterministic device design that may be incorporated into the present invention. FIG. 73B is a table of design parameters corresponding to FIG. 73A. FIG. 74 is a cross-sectional view of a magnetic separation device useful for the device of the present invention and the associated steps for cell isolation and subsequent offline analysis according to the present invention. FIG. 75 is a diagram schematically showing the manufacture and function provision of the magnetic separation device. The magnetized column can adjust the device after mounting. FIG. 76 is a diagram schematically showing the application of a magnetic separation device for capturing and releasing CD71 + cells from a complex mixture such as blood using a monoclonal antibody against the transferrin (CD71) receptor. FIG. 77 schematically illustrates application of a magnetic separation device for capturing and releasing CD71 + cells from a complex mixture such as blood using holotransferrin. Holotransferrin has a high iron content, is commercially available, and has a higher affinity constant and specificity for interaction with the CD71 receptor than the corresponding monoclonal antibody. FIG. 78 is a schematic diagram of a steep magnet. The magnet generates a magnetic field of 1.2 Tesla and a magnetic field gradient of about 3 Tesla / mm. FIG. 79A is a schematic view of a capillary disposed adjacent to the magnet of FIG. 78. FIG. 79B is a graph showing the magnetic field intensity by the magnet with respect to the position of the capillary. FIG. 79C is a photograph of red blood cells that have collected in separate areas after 10 minutes in a magnetic field. FIG. 80 is a photograph of a pellet of nucleated red blood cells (positive fraction) and a pellet of white blood cells (negative fraction) prepared from male umbilical cord blood. Nucleated cells are first extracted from blood using a deterministic horizontal separation device and treated with 50M sodium nitrite for 10 minutes. Nucleated cells are then passed through a magnetic column and nucleated red blood cells are retained in the column. The magnetic field strength in the column is about 1 Tesla, the magnetic field gradient is about 3000 Tesla / m, and the flow rate is about 0.4 mm / sec. It is. Leukocytes are washed off the column with Dulbecco's PBS buffer containing 1% BSA and 2 mM EDTA and collected as a negative fraction. Nucleated red blood cells are eluted from the column with the same buffer at a flow rate of 4 mm / s and collected as a positive fraction. FIG. 81 is a fluorescence image of nucleated red blood cells isolated from maternal blood using the method shown in FIG. Cells are stained by fluorescence in situ hybridization (FISH) method. The X chromosome is identified with an indigo green labeled probe for the alpha satellite region, while the Y chromosome is identified by red and green staining for the alpha satellite region and satellite III region, respectively. Nuclei are counterstained with DAPI (blue). FIG. 82 shows nucleated red blood cells at different stages of maturation isolated from maternal blood using the method shown in FIG. Cells are stained by light Giemsa staining. 83A and 83B are photomicrographs showing the results of concentration using the anti-CD71 antibody (A) and the method (B) shown in FIG. Sample A contained more than 200,000 nucleated cells obtained from 1 mL of blood, while sample B contained approximately 100 to 500 nucleated cells per mL of blood. The purity of nucleated red blood cells obtained by the method shown in FIG. 80 is about 1000 times better than the concentration method using an antibody. FIG. 84 is a diagram schematically showing three methods of the present invention. FIG. 85 is a diagram schematically showing an integrated device of the present invention. FIG. 86 is a flowchart for explaining the isolation of fetal erythrocyte nuclei. FIG. 87 is a graph schematically showing the state of cell lysis in a maternal blood sample. FIG. 88 is a diagram schematically illustrating a microfluidic method of concentrating target cells, preferably lysing the target cells in a concentrated sample. The sample is first concentrated by directing the target cells into the preferred channel based on size, and the target cells are then selectively lysed by controlling the residence time in the lysate. FIG. 89 is a diagram schematically illustrating a microfluidic method for isolating nuclei of target cells lysed from non-lysed whole cells that are not lysed based on size. Non-target cells are directed into the drainage and the nuclei are retained in the desired product stream. FIG. 90 is a flowchart showing another method for separating fetal cell nuclei from maternal leukocytes. FIG. 91 is a schematic diagram of a device of the present invention incorporating a substantially constant gap width, fluid supply interface, and fluid extraction interface. FIG. 92a is a schematic view of the manifold of the present invention. FIG. 92b is a photograph of the manifold of the present invention. FIG. 93 is a graph showing the ratio of living cells to time in a low osmotic pressure lysate. FIG. 94 is a graph showing hemolysis in whole blood versus time in lysis buffer. FIG. 95a is a table showing the yield of nuclei after cytospin using the Carnoy's fixative whole cell lysis method described herein. FIG. 95b is a fluorescence micrograph showing an example of the result of nuclear FISH method using Carnoy's fixative-mediated whole cell lysis method. Nuclei are FISH-treated for X (blue green), Y (green), and Y (red) and counterstained with DAPI. FIG. 96 is a flow chart detailing various options for cell and nucleus lysis. FIG. 97 is a schematic diagram of a typical magnetic concentration device. FIG. 98 is a photograph of a typical magnetic concentration device. FIG. 99 is a photomicrograph of red blood cells obtained using the method of the present invention. FIG. 100 is a photomicrograph of monocytes incorporating RBCs inside. FIG. 101 is a photomicrograph of fetal cells that are chromosome 21 trisomy enriched using the method of the present invention.

  The scale of each figure is not necessarily constant.

Claims (171)

A device for producing a sample in which a first cell or component thereof is concentrated relative to a second component, wherein the device comprises:
(A) a channel through which the first cell or component flows;
(B) a magnet that generates a magnetic field between 0.05 Tesla and 5.0 Tesla and a magnetic gradient between 100 Tesla / m and 1,000,000 Tesla / m in the channel;
A device characterized by comprising.   2. The device of claim 1 wherein the first cell or component is retained in the channel and the second component is not retained in the channel.   2. The device of claim 1 wherein the first cell or component is not retained in the channel and the second component is retained in the channel.   2. The device of claim 1, wherein the channel comprises first and second outlets, and the first cell or component thereof is directed to the first outlet, while the second component. Is directed to the second outlet.   The device of claim 1, further comprising an analysis module that concentrates the first cells or components based on size, shape, deformability, or affinity.   6. The device of claim 5, wherein the analysis module comprises the structure for deterministically deflecting particles having a hydraulic size larger than the critical size in a direction that is not parallel to the average flow direction in the structure. 6. The device of claim 5, comprising one channel, wherein the particle is the first cell or component, or the second component.   2. The device of claim 1, further comprising a reagent capable of changing a magnetic property of the first cell or component or the second component of the sample.   8. The device of claim 7, wherein the reagent alters the magnetic properties of a protein present in the first cell or component, or the second component.   9. The device of claim 8, wherein the protein comprises iron.   The device according to claim 9, wherein the protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.   9. The device of claim 8, wherein the reagent comprises sodium nitrite, carbon dioxide, oxygen, carbon monoxide, or nitrogen.   The device according to claim 1, wherein the first cell is a blood cell.   The device according to claim 1, wherein the first cell is a nucleated cell.   The device according to claim 1, wherein the first cell is an enucleated cell.   The device according to claim 1, wherein the first cell is an adult nucleated red blood cell.   2. The device of claim 1, wherein the first cell is a fetal nucleated red blood cell.   17. The device of claim 16, wherein the fetal nucleated red blood cells are from a fetus that is less than 10 weeks old.   2. The device according to claim 1, wherein the first cell is of a mammal, a bird, a reptile, or an amphibian.   The device of claim 1, wherein the component of the first cell is from the nucleus, perinuclear compartment, nuclear membrane, mitochondria, chloroplast, cell membrane, lipid, polysaccharide, protein, nucleic acid, virus particle, or ribosome. A device that is at least one selected from the group consisting of:   8. The device according to claim 7, wherein the reagent causes expression or overexpression of a magnetic protein in the first cell or component, or the second component.   21. The device of claim 20, wherein the reagent is capable of transfecting the first cell or the second component with a magnetically responsive protein.   8. The device of claim 7, wherein the reagent comprises magnetic particles that bind to the first cell or component or the second component or are taken up into the first cell. Device to use.   2. The device of claim 1, further comprising a pump capable of generating a flow rate that causes more than 50,000 cells per second or components thereof to flow into the channel.   2. The device of claim 1, wherein at least 90% of the first cell or component is retained within the device and at least 90% of the second component is not retained within the device. . A method for producing a sample in which a first cell or component thereof is enriched with respect to a second component, said method comprising:
(A) introducing a sample comprising said first cell or component into the device of claim 1;
(B) changing the path of the first cell or component or the second component in the sample based on magnetic properties relative to the other path, thereby concentrating the first cell or component; Producing said sample,
A method characterized by comprising.   26. The method of claim 25, wherein in step (a), the sample introduced into the device is enriched in the first cells or components relative to a third component.   27. The analysis module of claim 26, wherein prior to step (a), the first cell or component is concentrated based on size, shape, deformability, or affinity for the third component. And contacting the sample.   28. The method of claim 27, wherein the analysis module comprises a first structure that deterministically deflects particles having a hydraulic size larger than a critical size in a direction that is not parallel to the average flow direction in the structure. And wherein the particle is the first cell or component or the third component in the sample.   26. The method of claim 25, wherein the sample enriched in the first cells or components retains at least 70% of the first cells or components present in the sample. .   26. The method of claim 25, wherein the sample enriched in the first cells or components is concentrated at a ratio of 100 times.   26. The method of claim 25, wherein the sample enriched with the first cells is enriched at a ratio of at least 1000 times, and the first cells are nucleated red blood cells.   26. The method of claim 25, prior to step (b), contacting the sample with a reagent capable of changing the magnetic properties of the first cell or component or second component. A method further comprising:   33. The method of claim 32, wherein the reagent alters the magnetic properties of a protein present in the first cell or component or the second component.   34. The method of claim 33, wherein the protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.   34. The method of claim 33, wherein the reagent comprises sodium nitrite, carbon dioxide, or nitrogen.   26. The method of claim 25, wherein the first cell is a blood cell.   26. The method of claim 25, wherein the first cell is a nucleated cell.   26. The method of claim 25, wherein the first cell is an enucleated cell.   37. The method of claim 36, wherein the blood cell is an adult nucleated red blood cell.   40. The method of claim 39, wherein the adult nucleated red blood cells are used to monitor the diagnosis or treatment of erythroleukemia, severe beta thalassemia, Bart's hemoglobin, or immunohemolytic anemia. And how to.   37. The method of claim 36, wherein the blood cell is a fetal nucleated red blood cell.   42. The method of claim 41, wherein the fetal nucleated red blood cells are from a fetus that is less than 10 weeks old.   26. The method of claim 25, wherein the first cell is from a mammal, bird, reptile, or amphibian.   26. The method of claim 25, wherein the component of the first cell is a nucleus, perinuclear compartment, nuclear membrane, mitochondria, chloroplast, cell membrane, lipid, polysaccharide, protein, nucleic acid, virus particle, or ribosome. A method characterized by being.   33. The method of claim 32, wherein the reagent causes expression or overexpression of a magnetic protein in the first cell or component or the second component.   33. The method of claim 32, wherein the reagent binds to the first cell or component or the second component or is incorporated into the first cell or component or the second component. A method characterized by comprising particles.   26. The method of claim 25, wherein the sample enriched in the first cells or components is at least 90% of the first cells or components in the sample introduced in step (a), as well as steps. A method comprising less than 10% of the second component in the sample introduced in (a).   26. The method of claim 25, wherein more than 50,000 cells per second or components thereof flow into the channel. In a method of making a sample in which a first cell or component thereof is enriched relative to a second component, the method includes:
(A) contacting a sample comprising the first cell or component with a reagent that alters the magnetic properties of the protein expressed in the first cell or component or the second component in the sample; Producing a denatured sample;
(B) a magnetic field in which the denatured sample can have a magnet disposed relative to the channel to change the path of the first cell or component or the second component in the sample relative to the other path; Contacting the channel that generates a magnetic gradient, thereby producing the sample enriched in the first cells or components;
A method characterized by comprising.   50. The method of claim 49, wherein the sample comprising the first cell or component is enriched in the first cell or component relative to a third component before or after step (a). A method characterized by that.   51. The method of claim 50, prior to step (a), the sample is based on size, shape, deformability, or affinity of the first cell or component for the third component. Contacting the analysis module to be concentrated.   52. The method of claim 51, wherein the analysis module comprises a first structure that deterministically deflects particles having a hydraulic size larger than a critical size in a direction that is not parallel to the average flow direction in the structure. And wherein the particle is the first cell or component or the third component in the sample.   50. The method of claim 49, wherein the sample enriched in the first cells or components retains at least 70% of the first cells or components present in the sample. .   50. The method of claim 49, wherein the sample enriched in the first cells or components is enriched at a ratio of 100 times.   50. The method of claim 49, wherein the sample enriched in the first cells is enriched at a ratio of at least 1000 times, and the first cells are nucleated red blood cells.   50. The method of claim 49, wherein the reagent alters the magnetic properties of a protein present in the first cell or component or the second component.   57. The method of claim 56, wherein the protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.   57. The method of claim 56, wherein the reagent comprises sodium nitrite, carbon dioxide, or nitrogen.   50. The method of claim 49, wherein the first cell is a blood cell.   50. The method of claim 49, wherein the first cell is a nucleated cell.   50. The method of claim 49, wherein the first cell is an enucleated cell.   60. The method of claim 59, wherein the blood cell is an adult nucleated red blood cell.   63. The method of claim 62, wherein the adult nucleated red blood cells are used to monitor the diagnosis or treatment of erythroleukemia, severe beta thalassemia, Bart's hemoglobin, or immunohemolytic anemia. And how to.   60. The method of claim 59, wherein the blood cell is a fetal nucleated red blood cell.   65. The method of claim 64, wherein the fetal nucleated red blood cells are from a fetus that is less than 10 weeks old.   50. The method of claim 49, wherein the first cell is of a mammal, bird, reptile, or amphibian.   50. The method of claim 49, wherein the component of the first cell is a nucleus, perinuclear compartment, nuclear membrane, mitochondria, chloroplast, cell membrane, lipid, polysaccharide, protein, nucleic acid, virus particle, or ribosome. A method characterized by being.   50. The method of claim 49, wherein the reagent causes expression or overexpression of a magnetic protein in the first cell or component or the second component.   50. The method of claim 49, wherein the reagent binds to the first cell or component or the second component, or is incorporated into the first cell or component or the second component. A method characterized by comprising particles.   50. The method of claim 49, wherein the sample enriched in the first cells or components is at least 90% of the first cells or components in the sample contacted in step (a), as well as steps. A method comprising: comprising less than 10% of the second component in the sample contacted in (a).   50. The method of claim 49, wherein the magnet has a magnetic field in the channel between 0.05 Tesla and 5.0 Tesla and a magnetic gradient between 100 Tesla / m and 1,000,000 Tesla / m. A method characterized by generating.   50. The method of claim 49, wherein more than 50,000 cells or components thereof per second flow into the channel. A method of concentrating a first analyte from a fluid sample comprising a first analyte relative to second and third analytes in the sample, the method comprising:
(A) using a plurality of barrier structures to deflect the first analyte in a first direction and the second analyte in a second direction from the fluid sample based on hydraulic size; Performing a first concentration step of concentrating the first analyte;
(B) performing a second concentration step of concentrating the first analyte from the fluid sample based on the intrinsic or external magnetic properties of the first or third analyte;
A method characterized by comprising.   74. The method of claim 73, wherein the fluid sample is a blood sample.   74. The method of claim 73, wherein the fluid sample is a maternal blood sample.   74. The method of claim 73, wherein the one or more analytes are red blood cells.   74. The method of claim 73, wherein the one or more analytes are fetal nucleated red blood cells.   74. The method of claim 73, wherein the one or more analytes comprise fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome, respectively.   74. The method of claim 73, wherein the second concentration step comprises applying a magnetic field to the product of the first concentration step.   80. The method of claim 79, wherein the magnetic field attracts a first or third analyte.   80. The method of claim 79, wherein the magnetic field repels the first or third analyte.   80. The method of claim 79, wherein the magnetic field changes the path of the first analyte relative to a third analyte.   80. The method of claim 79, wherein the magnetic field is between 0.5 Tesla and 5.0 Tesla.   80. The method of claim 79, wherein the second concentration step further comprises the step of applying a magnetic gradient between 100 Tesla / m and 1,000,000 Tesla / m.   74. The method of claim 73, further comprising deoxygenating the product of the first concentration step. The method of claim 85, wherein said deoxygenation step is characterized by further comprising a product of the first concentration step CO, CO 2, _{N 2,} or contacting with NaNO ₂ .   74. The method of claim 73, further comprising the step of paramagnetizing the first or third analyte.   75. The method of claim 73, further comprising demagnetizing the first or third analyte.   75. The method of claim 73, wherein the first concentration step and the second concentration step are performed sequentially.   74. The method of claim 73, wherein the first concentration step comprises a plurality of concentration steps based on hydraulic sizes performed sequentially from one another.   74. The method of claim 73, wherein the first concentration step comprises a plurality of concentration steps based on hydraulic sizes performed in parallel with each other.   74. The method of claim 73, wherein the second concentration step comprises a plurality of concentration steps performed in parallel with each other.   75. The method of claim 73, wherein the first concentration step is performed while passing the sample.   75. The method of claim 73, wherein the second concentration step is performed while passing the sample.   74. The method of claim 73, wherein the second enrichment step is based on intrinsic magnetic properties.   74. The method of claim 73, wherein the second concentration step is based on external magnetic properties.   74. The method of claim 73, wherein more than 50,000 analytes are subjected to concentration per second. (A) One or more first analytes having a hydrodynamic size larger than the critical size in a first direction toward the first outlet and having a hydrodynamic size smaller than the critical size. A first module comprising an array of barrier structures that selectively deflects one or more second analytes in a second direction toward the second outlet;
(B) a second module comprising a channel for receiving one or more of the first analytes from the first outlet;
(C) a magnet that generates a magnetic field and a magnetic gradient in the channel to change the course of one or more of the first analytes;
A system characterized by comprising.   99. The system of claim 98, wherein the one or more second analytes comprise enucleated red blood cells.   99. The system of claim 98, wherein the one or more first analytes comprise nucleated red blood cells.   99. The system of claim 98, wherein the one or more first analytes comprise fetal nucleated red blood cells.   99. The system of claim 98, wherein the one or more first analytes comprise fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.   99. The system of claim 98, further comprising a reservoir containing an oxygen scavenger associated with the array of barrier structures or the channel.   99. The system of claim 98, further comprising a reservoir comprising a probe for specifically binding one or more of the first analyte or components thereof.   105. The system according to claim 104, wherein the probe is a nucleic acid probe or an antibody probe.   99. The system of claim 98, wherein the magnetic field is between 0.5 Tesla and 5.0 Tesla.   99. The system of claim 98, wherein the magnetic gradient is between 100 Tesla / m and 1,000,000 Tesla / m.   99. The system of claim 98, wherein the path of one or more of the first analytes changes based on intrinsic magnetic properties.   (A) One or more first analytes having a hydrodynamic size larger than the critical size in a first direction toward the first outlet, one hydrodynamic size smaller than the critical size A flow channel comprising a two-dimensional array of barrier structures that selectively deflects the second or more second analytes in a second direction toward the second outlet; (b) one or more flow channels; A magnet that generates a magnetic field and a magnetic gradient to change the path of the first analyte.   110. The system of claim 109, wherein the one or more first analytes are fetal nucleated red blood cells.   110. The system of claim 109, wherein the course of one or more of the first analytes changes based on the presence of hemoglobin.   110. The system of claim 109, wherein the magnetic field is between 0.5 Tesla and 5.0 Tesla.   110. The system of claim 109, wherein the magnetic gradient is between 100 Tesla / m and 1,000,000 Tesla / m. In a device for making a sample enriched for an analyte, said device:
(A) a first channel comprising the structure for deterministically deflecting particles having a hydrodynamic size larger than a critical size in a direction not parallel to an average flow direction in the structure, wherein the particles are the sample A first channel that is an analyte particle or a non-analyte particle;
(B) the reservoir fluidly connected to the effluent of the first channel through which the analyte flows into the reservoir, the reservoir changing a reagent that alters the magnetic properties of the analyte; A reservoir to provide;
A device characterized by comprising.   119. The device of claim 114, wherein the first channel is a microfluidic channel.   119. The device of claim 114, wherein the structure comprises an array of barrier structures forming a network of gaps, and the fluid flowing through the gap has an average flow direction of the main flux parallel to the average flow direction in the channel. A device characterized by being unequally divided into a main flux and a subflux so as not to become   117. The device of claim 116, wherein the array of barrier structures comprises first and second rows such that fluid flowing through the gaps in the first row is unevenly diverted to the two gaps in the second row. In addition, the device is characterized in that the second row is offset laterally with respect to the first row.   115. The device of claim 114, wherein the analyte has a hydraulic size that is greater than the critical size.   115. The device of claim 114, wherein the analyte has a hydrodynamic size that is smaller than the critical size.   119. The device of claim 114, further comprising a magnet capable of generating a magnetic field.   122. The device of claim 120, wherein the magnet comprises a region of a magnetic barrier structure disposed in a second channel.   122. The device of claim 121, wherein at least a portion of the magnetic barrier structure comprises a permanent magnet.   122. The device of claim 121, wherein at least a portion of the magnetic barrier structure comprises a non-permanent magnet.   122. The device of claim 121, wherein the barrier structures are arranged in a two-dimensional array.   122. The device of claim 121, wherein the second channel is a microfluidic channel.   119. The device of claim 114, wherein the reservoir further comprises a second channel comprising a magnet.   119. The device of claim 114, wherein the reagent changes an intrinsic magnetic property of one or more of the analytes.   128. The device of claim 127, wherein the reagent comprises sodium nitrite.   119. The device of claim 114, wherein the reagent binds to one or more of the analytes.   130. The device of claim 129, wherein the reagent comprises magnetic particles.   131. The device of claim 130, wherein the magnetic particles comprise an antibody or antigen binding fragment thereof.   132. The device of claim 131, wherein the antibody is anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, or anti-fetal hemoglobin.   130. The device of claim 129, wherein the reagent comprises holotransferrin. In a method of making a sample in which a first analyte is enriched with respect to a second analyte, the method includes:
(A) applying at least a portion of the sample to a device comprising the structure for deterministically deflecting particles having a hydraulic size larger than the critical size in a direction not parallel to the average flow direction in the structure; Thereby concentrating the first analyte to produce a second sample comprising the second analyte;
(B) combining the second sample with a reagent that alters the magnetic properties of the first analyte to produce a denatured first analyte;
(C) applying a magnetic field to the second sample, where the magnetic field provides a differential force to physically separate the denatured first analyte from the second analyte. Generating a sample that is enriched thereby concentrating the first analyte;
A method characterized by comprising.   135. The method of claim 134, wherein the reagent binds to the first analyte.   135. The method of claim 134, wherein the reagent alters an intrinsic magnetic property of the first analyte.   138. The method of claim 136, wherein the reagent comprises sodium nitrite.   135. The method of claim 134, wherein the reagent comprises magnetic particles that bind to the first analyte or are incorporated into the first analyte.   139. The method of claim 138, wherein the magnetic particle comprises an antibody or antigen-binding fragment thereof.   140. The method of claim 139, wherein the antibody is anti-CD71, anti-GPA, anti-antigen i, anti-CD45, anti-CD34, or anti-fetal hemoglobin.   135. The method of claim 134, wherein the analyte has a hydraulic size that is greater than the critical size.   135. The method of claim 134, wherein the analyte has a hydrodynamic size that is smaller than the critical size.   135. The method of claim 134, wherein the sample comprises a maternal blood sample.   135. The method of claim 134, wherein the first analyte is a cell, organelle, or virus.   145. The method of claim 144, wherein the cell is a bacterial cell, fetal cell, or blood cell.   145. The method of claim 145, wherein the blood cell is a fetal red blood cell.   145. The method of claim 144, wherein the organelle is a nucleus. A method for preparing a sample in which red blood cells are concentrated with respect to a second blood component, the method comprising:
(A) contacting a sample comprising red blood cells with a reagent that oxidizes iron to produce oxyhemoglobin;
(B) applying a magnetic field to the sample, wherein the red blood cells having oxyhemoglobin are attracted to the magnetic field stronger than the second blood component, thereby producing the sample in which the red blood cells are concentrated; ;
A method characterized by comprising.   148. The method of claim 148, wherein the red blood cells are fetal red blood cells.   149. The method of claim 149, wherein the second blood component is maternal blood cell.   148. The method of claim 148, wherein prior to step (a), the sample is enriched for the red blood cells.   152. The method of claim 151, wherein the enrichment deterministically deflects at least a portion of the sample in a direction that is not parallel to the average flow direction in the structure with particles having a hydraulic size larger than the critical size. A method characterized in that it is performed by applying to a device comprising said structure.   153. The method of claim 152, wherein fetal red blood cells are concentrated relative to maternal red blood cells. In a device for making a sample enriched in red blood cells, the device:
(A) an analytical device that concentrates the red blood cells based on size, shape, deformability, or affinity;
(B) a reservoir comprising a reagent that oxidizes iron, wherein the reagent enhances the magnetic responsiveness of the red blood cells;
A device characterized by comprising.   156. The device of claim 154, wherein the analytical device comprises a first structure that deterministically deflects particles having a hydraulic size greater than a critical size in a direction that is not parallel to the average flow direction in the structure. A device characterized by comprising a channel.   155. The device of claim 154, wherein the reagent is sodium nitrite. In a method for enriching a subpopulation of red blood cells, the method comprises:
(A) introducing a sample comprising red blood cells into a column and applying a magnetic field to the column;
(B) Based on magnetic properties, the path of the first type of red blood cells in the sample is changed relative to the second type of red blood cells, where the first type of red blood cells are the first type of red blood cells. Being attracted to the magnetic field more strongly than two types, thereby concentrating the red blood cell subpopulation;
A method characterized by comprising.   158. The method of claim 157, wherein the first type of red blood cell is a positive erythroblast.   158. The method of claim 157, wherein the first type of red blood cell is a mature red blood cell.   158. The method of claim 157, wherein the second type of red blood cells are polychromatic erythroblasts.   158. The method of claim 157, wherein the second type of red blood cell is a positively stained erythroblast. In a method for concentrating a population of cells harboring magnetically sensitive particles, the method includes:
(A) introducing a sample comprising cells into a column and applying a magnetic field to the column;
(B) based on the magnetic properties, changing the path of the cells containing the magnetically sensitive particles in the sample relative to the second type of cells in the sample, thereby changing the cell population Concentrating;
A method characterized by comprising.   163. The method of claim 162, wherein the magnetically susceptible particles are red blood cells.   164. The method of claim 162, wherein the magnetically susceptible particles are magnetotactic bacteria.   163. The method of claim 162, wherein the magnetically susceptible particles comprise magnetite or greigite.   163. The method of claim 162, wherein the cells containing the magnetically susceptible particles are monocytes or macrophages.   164. The method of claim 162, wherein the cells harboring the magnetically susceptible particles are used to diagnose familial hemophagocytic histiocytosis, acute monocytic leukemia, or lymphoma or to monitor its treatment. A method characterized by that. In a method for removing red blood cells and white blood cells of a blood sample, the method comprises:
(A) introducing a sample comprising blood cells into a column and applying a magnetic field to the column;
(B) contacting the sample with magnetically sensitive particles that bind to white blood cells before, during, or after step (a);
(C) changing the path of red blood cells and white blood cells in the sample to a third type of cell based on magnetic properties, thereby removing the red blood cells and white blood cells of the sample;
A method characterized by comprising.   169. The method of claim 168, wherein the third type of cell comprises a stem cell.   168. The method of claim 168, wherein the blood sample is a cord blood sample.   169. The method of claim 168, wherein the magnetically sensitive reagent comprises an anti-CD45 or anti-CD15 antibody.

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