CN101305087A - Device and method for magnetic enrichment of cells and other particles - Google Patents

Abstract

The present invention relates to devices and methods for the enrichment of cells and other desired analytes by employing a magnetic field, alone or in conjunction with size-based separation. The devices and methods may be advantageously employed to enrich for rare cells, e.g., fetal cells or epithelial cells, present in a sample, e.g., maternal blood.

Description

Be used for magnetic enrichment of cells and other particulate apparatus and method

Background technology

The present invention relates to cellular segregation, medical diagnosis and microfluidic device.The diagnosis or the information of environmental correclation often are present in the sample, detect but amount is too low less than.Therefore, often use various enrichments or amplification method to increase the detectability of such information.

For cell, can use different flow cytometries and cell sorting method, but these technology are usually used big and equipment unit costliness, need a large amount of samples and technological operation personnel.These cytolytic dose instrument and sorter use and resemble electrostatic deflection, centrifugal, fluorescence-activated cell sorting (FACS) and magnetic activating cells sorting (MACS) to realize cellular segregation.The abundant enriched sample of these methods Chang Buneng is to allow the rare component of analytic sample.In addition, these technology can cause so unacceptable loss of rare component, for example separate or degraded by the inefficiency of component.

The new device and the method that therefore, need be used for enriched sample.

Summary of the invention

Usually, the present invention relates to allow usually to use the apparatus and method of magnetite gathering cell and interested other analytes in conjunction with another kind of separation parameter for example size, shape, deformability or avidity.Preferably, interested analyte separates based on intrinsic magnetic, and described intrinsic magnetic can as described hereinly change.

Therefore, the present invention relates to a kind of device that is used to generate the enriched sample of enrichment first cell or its component (with respect to second component), it comprises: the passage that described first cell or component stream are crossed, and the magnetic field and the 100 tesla/m to 1 that in described passage, produce 0.05 to 5.0 tesla, the magnet of the field gradient of 000,000 tesla/m.Described first cell or component can be retained in the described passage, and described second component can not be retained in the described passage, or vice versa.Described passage can comprise first outlet and second outlet, and wherein said first cell or its component import described first outlet, and described second component imports described second outlet.Described device also can comprise producing and flows into the pump of described passage greater than the flow velocity of 50,000 cells or its component p.s..

Described device also can comprise the analytical unit based on described first cell of size, shape, deformability or avidity enrichment or component.Described analytical unit comprises the first channel that for example has following structure: described structure is with the direction determinacy deflected stream mechanics size that is not parallel to mean flow direction in the described structure particle greater than critical size.Described structure can comprise the obstacle array that forms the gap network, and the fluid by described gap is divided into main flow and time stream unequally therein, makes the mean direction of described main flow be not parallel to the mean direction of the fluid flow in the described passage.Described obstacle array can comprise first row and second row, and wherein said second row's side is put described first discharging, makes the fluid that flows through the described first row gap divide two gaps into described second row unequally.

Described device also can comprise the reagent of the magnetic that can change described first cell or the component or second component.For example, described reagent changes the magnetic that is present in for example ferruginous protein of protein (for example foetal haemoglobin, adult haemoglobin, methemoglobin, myohaemoglobin or cytopigment) in described first cell or component or described second component.Exemplary agents comprises Sodium Nitrite, carbonic acid gas, oxygen, carbon monoxide and nitrogen.Described reagent also can cause having the protein expression of magnetic or cross and express in described first cell or component or described second component.For example, described reagent can use described first cell of magnetic response protein transfection or described second component.Described reagent also can comprise the magnetic-particle that is bonded to or mixes described first cell or component or described second component.

Described first cell be for example hemocyte (as adult's erythroblast, or fetal nucleated red blood, for example from less than 10 week age fetus), another kind of karyocyte or cytode.Described first cell can be mammals, birds, reptilia or batrachians.The exemplary compositions of described first cell comprises nucleus, examines all compartments, nuclear membrane, plastosome, chloroplast(id) or cytolemma, lipid, polysaccharide, protein, nucleic acid, virion and rrna.

In a preferred embodiment, at least 90% described first cell or component are retained in the described device, and at least 90% described second component is not retained in the described device.

On the other hand, device of the present invention is used to produce the sample of enrichment first cell or its component (with respect to second component), introduce passage by the sample that will comprise first cell or component, and make the path of described first cell or component or described second component change based on magnetic, thereby produce the sample of described first cell of enrichment or component with respect to another component.The described sample of introducing described device can be with respect to the 3rd component enrichment first cell or component.For example, described sample can with based on size, shape, deformability, cleaved ability, mark or avidity and contact with respect to the analytical unit of the 3rd component enrichment first cell or component.The exemplary analysis assembly comprises the first channel with following structure: described structure is with the direction determinacy deflected stream mechanics size of the mean flow direction that the is not parallel to described structure particle greater than critical size, and wherein said particle is first cell or the component or the 3rd component of sample.Enrichment the sample of first cell or component at least 70% first cell or the component that exist in can keeping sample.Enrichment first cell or component example enrichment for example 100 times.Described method also comprises makes sample contact with the reagent of the magnetic that can change first cell or component or second component.Enrichment the sample of first cell or component can be included in the sample of introducing before the enrichment before at least 90% first cell or component and the enrichment and be less than 10% second component in the sample.Exemplary agents, first cell, its component, second component, purity and flow velocity are described at this paper.

The invention still further relates to a kind of alternative method that has produced with respect to the second component enrichment sample of first cell or its component by following step: the sample that comprises first cell or component is potentially contacted, the magnetic of expressed protein in first cell of described reagent change sample or the component or second component and mutagenic sample with reagent described herein; The sample of change is contacted with the passage with magnet, described magnet is placed with respect to passage and is produced and can change first cell or component or second component magnetic field and the field gradient with respect to the path of another component, thus the sample of first cell that produced enrichment or component.In certain embodiments, before making sample and magnetic field contact, sample with respect to the 3rd component enrichment first cell or component.For example, described sample can with based on size, shape, deformability, cleaved ability, mark or avidity and the analytical unit contact of relative the 3rd component enrichment first cell or component.The exemplary analysis assembly comprises the first channel with following structure: described structure is with the direction determinacy deflected stream mechanics size of the mean flow direction that the is not parallel to described structure particle greater than critical size, and wherein said particle is first cell or the component or the 3rd component of sample.Example results is described at this paper.But enrichment at least 70% first cell or the component that exists in the sample keeping sample of first cell or component.Enrichment first cell or component example enrichment for example at least 100 times or 1000 times.Enrichment the sample of first cell or component can be included in the sample of introducing before the enrichment before at least 90% first cell or component and the enrichment and be less than 10% second component in the sample.Exemplary agents, first cell, its component, second component, purity and flow velocity are described at this paper.

On the other hand, the present invention relates to a kind of with respect to second analyte and the 3rd enrichment of analytes from fluid sample (blood sample for example, the method of first analyte maternal blood sample for example), described method is to instruct first analyte and instruct first enriching step of a plurality of obstacle enrichments of second analyte from first analyte of fluid sample with second direction with first direction by implementing to use based on the hydromeehanics size, and implements based on the inherent or external magnetite gathering of first analyte or the 3rd analyte second enriching step from first analyte of fluid sample.Exemplary first analyte is a cell, and is as described herein.Second enriching step can comprise the product that magnetic field is put on first enriching step.Magnetic field can attract or repel first analyte or the 3rd analyte.Usually, magnetic field changes the path of first analyte with respect to the 3rd analyte.Described method also comprises the step of the magnetic that changes first enriched product, for example passes through deoxidation.The deoxidation step can comprise product and CO, the CO that makes first enriching step₂, N₂Or NaNO₂Contact.Described method also can comprise paramagnetic (paramagnetizing) or degaussing (diamagnetizing) first analyte or the 3rd analyte.First enriching step and second enriching step can occur in sequence.In certain embodiments, first enriching step or second enriching step comprise a plurality of orders or a plurality of enriching step of the generation that is parallel to each other.First enriching step or second enriching step take place in the process of sample flow warp.Second enriching step can be based on inherent or external magnetic.Preferably, stand enrichment greater than 50,000 analyte p.s..Example magnetic field and field gradient are described at this paper.

The invention still further relates to a kind of system, described system comprises first assembly, second assembly and magnet, wherein first assembly has the obstacle array, this obstacle array instructs to have greater than one or more first analytes of the hydromeehanics size of critical size with the second direction selectivity towards second outlet with the first direction selectivity towards first outlet and instructs one or more second analytes that have greater than the hydromeehanics size of critical size, described second assembly has the passage that is used to receive from first analyte of first outlet, and described magnet produces magnetic field and the field gradient that changes the first analyte path in passage.

The invention still further relates to a kind of system, described system comprises circulation passage and the magnet with two-dimentional obstacle array, wherein said obstacle array instructs to have greater than one or more first analytes of the hydromeehanics size of critical size with the second direction selectivity towards second outlet with the first direction selectivity towards first outlet and instructs one or more second analytes that have greater than the hydromeehanics size of critical size, and wherein magnet produces magnetic field and the field gradient that changes the first analyte path.

First analyte is an erythroblast for example, and fetal nucleated red blood for example, second analyte are erythroplastids for example.Adult's erythroblast can be used for the diagnosis and the treatment of multiple disease, and is as described herein.First analyte comprises for example foetal haemoglobin, adult haemoglobin, methemoglobin, myohaemoglobin or cytopigment.Described system also comprises the bank that is coupled to obstacle array or passage, and described bank contains other reagent that Oxygen Scavenger maybe can change magnetic.Described system also can comprise and contains specificity in conjunction with the probe of first analyte or its component bank of nucleic acid probe or antibody probe for example.The path of first analyte changes based on for example inherence or external magnetic.The exemplary magnetic intensity of field that is used for described system is between 0.5 tesla and 5.0 teslas, and the exemplary magnetic field gradient is in 100 teslas/m and 1,000, between 000 tesla/m.Described system also can comprise can produce the p.s. flow channel greater than the pump of the flow velocity of 50,000 cells or its component.

On the other hand, the invention provides the device of the sample that has been used to produce enrichment analyte, described device comprises first channel (for example microfluidic channel) and bank, described first channel comprises following structure: with the direction determinacy deflected stream mechanics size of the mean flow direction that the is not parallel to described structure particulate structure greater than critical size, wherein said particle is the non-analyte component of analyte particles or sample; Described bank fluid is coupled to the outlet of first channel, and described analyte enters bank by the outlet of described first channel, and described bank can comprise the reagent that changes analyte magnetic.Described structure comprises the obstacle array that for example forms the gap network, and wherein the fluid by described gap is divided into main flow and time stream unequally, makes size follow main flow greater than the particle of critical size, and size is followed time stream less than the particle of critical size.Described obstacle array can comprise first row and second row, and wherein said second row's side is put described first discharging, makes the fluid that flows through the described first row gap be divided to go into two gaps of described second row unequally.Required analyte can have the hydromeehanics size of the critical size of being greater than or less than.Described device can comprise the magnet that can produce magnetic field, and also can comprise the magnetic obstacle that is disposed in the second passage (for example microfluidic channel) (as comprise the obstacle of permanent magnet or comprise the obstacle of impermanency magnet).The magnetic obstacle can be the two-dimensional array ordering.The bank of described device also can comprise the second passage that comprises magnet.Reagent (for example Sodium Nitrite) can change the inherent magnetic of one or more analytes.In one embodiment, for example full Transferrins,iron complexes of reagent or magnetic-particle can be in conjunction with one or more analytes.Magnetic-particle also can comprise antibody (antibody of for example anti-CD71, anti-CD36, anti-CD45, anti-GPA, antigen i, anti-CD34, anti-foetal haemoglobin, anti-EpCAM, anti-E-cadherin or anti-Muc-1) or its Fab.

The present invention also provides a kind of method that is used to have generated with respect to second enrichment of analytes sample of first analyte, described method comprises: with the described sample application of at least a portion in the device that comprises following structure, promptly with the direction determinacy deflected stream mechanics size of the mean flow direction that is not parallel to described structure greater than the described structure of the particulate of critical size, thereby first analyte that produced enrichment and comprise second sample of second analyte; The reagent of mutagenic first analyte combines with the magnetic that changes described first analyte to make described second sample; And magnetic field put on described second sample, and magnetic field produces first analyte and second analyte that difference power comes physical sepn to change therein, thus the sample of first analyte that produced enrichment.Described reagent can be in conjunction with described first analyte.In another embodiment, described reagent (for example Sodium Nitrite) can change the inherent magnetic of described first analyte.In another embodiment, described reagent can comprise the magnetic-particle that is bonded to or mixes described first analyte.Described magnetic-particle can comprise antibody (for example anti-CD71, anti-GPA, antigen i, anti-CD45, anti-CD34, anti-foetal haemoglobin, anti-EpCAM, anti-E-cadherin or anti-Muc-1) or its Fab.Described analyte can have the hydromeehanics size of the critical size of being greater than or less than.Described sample can be the maternal blood sample.Described first analyte can be cell (for example bacterial cell, fetal cell or red corpuscle for example fetal erythrocyte), organoid (for example nucleus) or virus.

The method of erythrocytic sample that the present invention also provides relative second red blood cell component of a kind of generation (for example parent red corpuscle) enrichment, described method comprises: the sample that comprises red corpuscle (for example fetal erythrocyte) is contacted with the reagent that makes the iron oxidation and produce oxygenated haemoglobin; And magnetic field put on sample, the red corpuscle that has oxygenated haemoglobin therein is attracted to magnetic field with the degree greater than second red blood cell component, thereby produces the sample of erythrocyte enrichment.Described method also can be included in the step of implementing the erythrocytic sample of enrichment before the contact procedure (for example the fetal erythrocyte of enrichment with respect to the parent red corpuscle and enrichment), for example by with the sample application of at least a portion in the device that comprises following structure, promptly have particle greater than the hydromeehanics size of critical size with the direction determinacy deflection of the mean flow direction that is not parallel to described structure.

On the other hand, the invention provides a kind of device that is used to generate enrichment erythrocytic sample, described device comprises: based on size, shape, deformability or the erythrocytic analytical equipment of avidity enrichment; And the bank that comprises the reagent that makes the iron oxidation, wherein said reagent (for example Sodium Nitrite) increases erythrocytic magnetic reactivity.Described analytical equipment can comprise the first channel that comprises following structure: promptly have particulate structure greater than the hydromeehanics size of critical size with the direction determinacy deflection of the mean flow direction that is not parallel to described structure.

The present invention also provides a kind of reagent, described reagent comprises the magnetic-particle of the multiple bound fraction that is coupled to one or more selective binding GPA, foetal haemoglobin, antigen-i, antigen-I, CD34, CD45, CD15, CD71, EGFR or EpCAm (for example antibody, as monoclonal antibody).

These reagent can be applicable to separate from cell mixture the method for one or more interested cells, described method comprises makes described cell mixture combine with reagent, and the described cell mixture of incubation and described reagent are enough to make time of interested one or more cells in the described bound fraction selective binding mixture, and magnetic field put on described mixture, thereby will be in conjunction with the cellular segregation of the cell and the debond magnetic-particle of magnetic-particle.Described method also can comprise the step of one or more interested cells in the enrichment of cell mixture.Described enriching step can comprise one or more cells that use the obstacle array implement to be uninterested cell based on the separation or the selective splitting of size.

In other respects, the present invention relates to a kind of method of enrichment erythroid cells subgroup, described method is to introduce post and magnetic field is put on described post by the sample that will comprise erythroid cells, kind of the erythroid cells of winning is changed based on magnetic with respect to the path of second kind of erythroid cells, and wherein first kind of erythroid cells is subjected to magnetic field suction greater than second kind of erythroid cells.Described first kind of erythroid cells is for example mature erythrocyte or eosinophilic normoblast, and described second kind of erythroid cells is for example eosinophilic normoblast or rubricyte.

The invention still further relates to a kind of enrichment and have the method for the magnetic susceptible particulate cell mass of including in, described method is to introduce post and magnetic field is put on described post by the sample that will contain cell, and makes the path that has the magnetic susceptible particulate cell of including in the sample be changed with respect to second kind of cell based on magnetic.Magnetic susceptible particle is for example red corpuscle or magnetotactic bacteria (magnetotactic bacteria).Magnetic susceptible particle can comprise magnetite (magnetite) or sulphur iron-stone (greigite).Has the magnetic susceptible particulate cell of including in and is for example monocyte or scavenger cell.Described method can be used for familial hemophagocytic histiocytosis, acute monocytic leukemia and lymphadenomatous diagnosis or treatment monitoring.

The invention still further relates to red corpuscle and leukocytic method in a kind of removing sample (for example Cord blood), described method is introduced post and magnetic field is put on post by comprising erythrocytic sample, before introducing, in the process or described sample is contacted with magnetic susceptible reagent in conjunction with white corpuscle (for example through anti-CD45 antibody or anti-CD15 antibody), make that red corpuscle and the leukocytic path in the sample is changed with respect to the third cell (as stem cell) based on magnetic.

" analyte " is meant molecule, other chemicals, for example iron, or particle.Exemplary analyte comprises cell, virus, nucleic acid, protein, carbohydrate and little organic molecule.

" biological particles " is meant any biogenetic derivation thing by the water insoluble medium of time range of sample collecting, preparation, storage and analysis.The example comprises cell, granulosa cell component, virus and the mixture that comprises protein, lipid, nucleic acid and carbohydrate.

" biological sample " is meant any biogenetic derivation sample or contains or the potential any sample that contains biological particles.Preferred biological sample is a cell sample.

Be meant for " blood component " and any component of whole blood comprise host's red corpuscle, white corpuscle and thrombocyte.The blood component also comprises plasma component, for example protein, lipid, nucleic acid and carbohydrate and for example because current or pregnancy in the past, organ transplantation, disease or infection can be present in any other cell in the blood.

" cell sample " is meant the sample that contains cell or its component.Such sample comprises that the part of naturally occurring liquid (for example blood, lymph liquid, cerebrospinal fluid, urine, uterine neck irrigating solution (cervical lavage) and water sample), these liquid and cell are introduced into liquid (for example tissue sample of substratum and liquefaction) wherein.This term also comprises lysate.

" catch part " and be meant analyte bonded compound part.Catching part can be the compound that is coupled to the surface or constitutes the material on surface.Exemplary acquisition partly comprises antibody, oligonucleotide or polynucleotide, nucleic acid, other protein, synthetic polymkeric substance and carbohydrate.

" passage " is meant the gap that fluid can flow through.Passage can be the band of the hydrophilic pattern on kapillary, conduit or the other hydrophobic surface that limits aqueous fluid therein.

" component " of cell is meant can be to any component of small part isolated cells when lysis.Cellular component can be organoid (for example nucleus, examine all compartments, nuclear membrane, plastosome, chloroplast(id) or cytolemma), polymkeric substance or molecular complex (for example lipid, polysaccharide, protein (film, through film or cytoplasmic), nucleic acid (natural, therapeutic or pathogenic), virion or rrna) or other molecules (for example hormone, ion, cofactor or medicine)." component " of cell sample is meant the cell subclass that contains in the sample.

" sample of enrichment " is meant the sample that contains analyte that is processed into respect to the relative quantity that is present in other analytes increase analytes in the sample usually.For example, can be by increasing interested analyte at least 10%, 25%, 50%, 75%, 100% or at least 1000,10,000,100,000 or 1,000,000 times and the sample of enrichment.

" sample of removing " is meant and contains the sample that contains analyte that is processed into respect to the relative quantity that is present in other analytes minimizing analytes in the sample usually.For example, can be by reducing interested analyte at least 5%, 10%, 25%, 50%, 75%, 90%, 95%, 97%, 98%, 99% or even 100% removing sample.

" exchange buffering liquid " in the context of sample (for example cell sample) is meant and is different from the initial matrix that suspends of sample wherein and one or more components matrix to be replaced of sample.

" the external magnetic " of analyte is meant for the endogenic magnetic of analyte right and wrong.

" the extraction border of flowing " is meant and is designed to remove the fluidic border from array.

" supply boundary flows " is meant the border that is designed to fluid is added into array.

" gap " is meant the opening that fluid and/or particle can flow through.For example, the gap can be kapillary, the interval between fluid two obstacles that can flow through wherein, or the hydrophilic pattern on the confined other hydrophobic surface of aqueous fluid wherein.In an embodiment preferred of the present invention, the gap network is limited by the obstacle array.In this embodiment, the gap is the interval between the adjacent barrier.In a preferred embodiment, the gap network is by the obstacle Array Construction on the stromal surface.

" hydromeehanics size " is meant the effective dimensions when particle and fluid, obstacle (for example post) or other particles interact.Described obstacle or other particles can be microfluidic structures.It is used as the generic term of deformability in particle volume, shape and the fluid.

" activate " activation that is meant second messenger's approach in the cell, it causes transcription factor activation or kinases or other pathways metabolisms to activate.By activating the change that also can cause the acceptor transportation in the cell of regulating outside membrane antigen.

" the intrinsic magnetic " of analyte is meant that for analyte be endogenic magnetic.Intrinsic magnetic can be present in when measuring beginning, or it can be induced by suitable reagent in analyte.Exemplary intrinsic magnetic comprises those that ferritin gives that contain of cell expressing.

" labelled reagent " is meant can bound analyte, is included into, or otherwise is adsorbed and is detected the reagent of (for example by shape, form, color, fluorescence, luminous, phosphorescence, absorption, magnetic or radioactive emission).

" metabolism group " is the keeping of the participation metabolic reaction except protein and nucleic acid and cell in the phalangeal cell, growth or the required compound group of normal function.

" microfluid " represents that the unidimensional size is less than 1mm at least.

" obstacle " is meant mobile encumbrance in the passage, for example outstanding from a surface.For example, but the obstacle phalangeal process for the hydrophobic barrier of post on the base substrate or aqueous fluid.In certain embodiments, described obstacle can have the part perviousness.For example, obstacle can be the post that porous material is made, and wherein, described hole allows water component to see through, but too little so that can not enter for separated particle.

" contraction reagent " is meant the reagent that reduces granule fluid mechanics size.Shrinking reagent can be by reducing particle volume, increases its deformability or changes its shape and work.

" expansion reagent " is meant the reagent that increases particulate hydromeehanics size.Expansion reagent can be by increasing particle volume, reduces its deformability or change its shape and work.

" in fact greater than " is meant at least greatly to 2 times, 3 times, 5 times, 10 times, 25 times, 50 times or even 100 times.

" in fact less than " is meant to when young to 1/2,1/3,1/5,1/10,1/25,1/50 or even 1/100.

From following description and claim, other feature and advantage will be apparent.

The accompanying drawing summary

Figure 1A-1E is based on the synoptic diagram that the determinacy sidesway comes the array of isolated cell: (A) described continue after row sidesway; (B) described flow through the gap fluid how continue after separated unequally around row's the obstacle; (C) described have greater than the analyte of the hydromeehanics size of critical size how in device by sidesway; (D) array of cylindrical obstacle has been described; And the array of (E) having described oval obstacle.

Fig. 2 describes by the separate fluid unequally of obstacle peripheral clearance in arrange the back.

Fig. 3 is the synoptic diagram how critical size depends on flow pattern, and flow pattern is parabola shaped in this embodiment.

Fig. 4 be shape how the impact analysis thing by the figure that moves of device.

Fig. 5 be deformability how the impact analysis thing by the figure that moves of device.

Fig. 6 is the synoptic diagram of determinacy sidesway.The analyte that has greater than the hydromeehanics size of critical size moves to the edge of array, and the analyte with hydromeehanics size of subcritical size does not have sidesway by device.

Fig. 7 is a syllogic determinacy schematic representation of apparatus.

Fig. 8 is the overall dimension of Fig. 7 device and the synoptic diagram of holding back size.

Fig. 9 is the synoptic diagram of by-pass channel.

Figure 10 is the synoptic diagram of by-pass channel.

Figure 11 is the syllogic determinacy schematic representation of apparatus with common bypass passage.

Figure 12 is the syllogic diad determinacy schematic representation of apparatus with common bypass passage.

Figure 13 is the syllogic determinacy schematic representation of apparatus with common bypass passage, is constant basically by flowing of device wherein.

Figure 14 is the syllogic diad determinacy schematic representation of apparatus with common bypass passage, is constant basically by flowing of device wherein.

Figure 15 is the syllogic determinacy schematic representation of apparatus with common bypass passage, and wherein the fluid resistance in by-pass channel and the adjacent segment is constant basically.

Figure 16 is the syllogic diad determinacy schematic representation of apparatus with common bypass passage, and wherein the fluid resistance in by-pass channel and the adjacent segment is constant basically.

Figure 17 is the syllogic determinacy schematic representation of apparatus with two by-pass channels that separate.

Figure 18 is the syllogic determinacy schematic representation of apparatus for two by-pass channels that separate with any configuration.

Figure 19 is the syllogic diad determinacy schematic representation of apparatus with three by-pass channels that separate.

Figure 20 is the syllogic determinacy schematic representation of apparatus with two by-pass channels that separate, and is constant basically by flowing of each section wherein.

Figure 21 is the syllogic diad determinacy schematic representation of apparatus with three by-pass channels that separate, and is constant basically by flowing of each section wherein.

Figure 22 flows to extract the synoptic diagram on border.

Figure 23 is the synoptic diagram of mobile supply boundary.

Figure 24 is the synoptic diagram that comprises the mobile supply boundary of by-pass channel.

Figure 25 is the synoptic diagram of two mobile supply boundaries of central by-pass channel side.

Figure 26 has four schematic representation of apparatus as the passage of (on-chip) resistance to flow device on the wafer.

Figure 27 and 28 is synoptic diagram that resistance gauge influences mobile two fluidic relative widths in the device on the wafer.

Figure 29 is the diad schematic representation of apparatus with common inlet of two external regions.

Figure 30 A is the synoptic diagram of a plurality of arrays on the device.Figure 30 B is the synoptic diagram that has many arrays of common inlet and product outlet on the device.

Fig. 31 is the multi-stage type schematic representation of apparatus with little footprint.

Figure 32 is that blood passes through schematic representation of apparatus.

Figure 33 is a graphic representation of describing the hydromeehanics distribution of sizes of hemocyte.

Figure 34 A-34D is the synoptic diagram that analyte moves to damping fluid from sample in single hop (A), three sections (B), diad (C) or syllogic diad (D) determinacy device.

Figure 35 A is used for particle is moved to damping fluid to produce the two-section type determinacy schematic representation of apparatus of three kinds of products from blood.Figure 35 B is two sections overall dimension and the profile of holding back size.Figure 35 C is the profile of the composition of three kinds of products.

Figure 36 is optional two-section type determinacy schematic representation of apparatus, and wherein each section has by-pass channel.

Figure 37 is that the fluid channel is used to connect two sections synoptic diagram in the device.

The fluid channel is used to connect two sections synoptic diagram in Figure 38 device, and wherein said two sections are configured to micropodia mark array.

Figure 39 A has the two-section type determinacy schematic representation of apparatus of acceptance from the by-pass channel of two sections output.

Figure 39 B is this profile that installs the size range of attainable product.

Figure 40 is the two-section type determinacy schematic representation of apparatus that optionally has by-pass channel, and adjacent each section of wherein said by-pass channel side and stream inject identical outlet.

Figure 41 is used for the determinacy schematic representation of apparatus that particle moves and changes in proper order.

Figure 42 A is the photo that can incorporate the determinacy device of apparatus of the present invention into.Figure 42 B-42E has described and has been used to assemble the covert that can incorporate device of the present invention into.Figure 42 F is a series of photos that contain the device of blood and damping fluid.

Figure 43 A-43F is blood sample and refuse (damping fluid, blood plasma, red corpuscle and thrombocyte) and product (damping fluid and karyocyte) the typical histogram partly that the device of blood analyser generation Figure 42 produces.

Figure 44 A-44D has described the covert that is used to assemble the determinacy device that can incorporate apparatus of the present invention into.

Figure 45 A-45D has described the covert that is used to assemble the determinacy device that can incorporate apparatus of the present invention into.

Figure 46 A is the Photomicrograph with the sample of fetal erythrocyte enrichment.Figure 46 B is the Photomicrograph of parent red corpuscle refuse.

Figure 47 is a series of Photomicrographs (blueness=nucleus, redness=X chromosome, green=Y chromosome) that show male fetal cell positive identification.

Figure 48 is a series of Photomicrographs that show the positive identification of sex chromosome and trisomy 21.

Figure 49 A-49D has described the covert that is used to assemble the determinacy device that can incorporate apparatus of the present invention into.

Figure 50 A-50G is the electron photomicrograph of the device of Figure 49.

Figure 51 A-51D has described the covert that uses the determinacy device that can incorporate apparatus of the present invention in the device.

Figure 52 A-52F is the electron photomicrograph of the device of Figure 51.

Figure 53 A-53F is the electron photomicrograph of the device of Figure 45.

Figure 54 A-54D describes the covert that example is used to assemble the determinacy device that can incorporate apparatus of the present invention into.

Figure 55 A-55S is the electron photomicrograph of the device of Figure 54.

Figure 56 A-56C is the electron photomicrograph of the device of Figure 44.

Figure 57 A can incorporate the determinacy device of apparatus of the present invention and the synoptic diagram of operation thereof into.Figure 57 B has described the device of Figure 57 A and the further synoptic diagram of this device.

Figure 58 A has described two different configurations that are used for two determinacy devices are linked together with 58B.In Figure 58 A, shown cascade configuration, theinlet 1 of one of them device is connected with the sample inlet of the twoth device, in Figure 58 B, has shown that the band wildcard puts, and theinlet 2 of one of them device is connected with the sample inlet of the twoth device.

Figure 59 has described the strong method that adds of apart, wherein uses immune affine pearl labels targets cell.

Figure 60 has described and a kind ofly has been used to carry out the size classification and can incorporates device of the present invention into from conjunction with the labelled reagent separated free labelled reagent method of antibody for example by using.

Figure 61 has described the method that Figure 60 shows.In this case, non-target cell can with the target cell copurification, but these non-target cells do not disturb target cell quantitatively.

Figure 62 has described and a kind ofly has been used for from the mixture separation maxicell and produces the method for the concentrating sample of these cells.

Figure 63 has described a kind of method that is used for cracking apparatus of the present invention inner cell and separates intact cell from organoid and other cellular components.

Figure 64 has described with cascade configuration and has arranged and be used to carry out size classification and the application of the invention device two kinds of devices from the labelled reagent of bonded labelled reagent separated free.

Figure 65 has described with cascade configuration and has arranged and be used to carry out size classification and the application of the invention device two kinds of devices from the labelled reagent of bonded labelled reagent separated free.In the figure, phage and non-antibody is used for combination and detection.Figure 66 has described two kinds of devices arranging in the band wildcard is put.

Figure 67 is the graphic representation with respect to the cell counting convection cell cell dia of microfluidic separation of normal whole blood.

Figure 68 is Coulter " A^c-T diff " one group of histogram of clinical haemanalysis the instrument input, product and the refuse sample that produce.X-axis is described the cell volume that femtomole is represented.

Figure 69 is to use the product of the fetal blood that the cell enrichment assembly handles and a pair of typical Photomicrograph of waste streams, has shown karyocyte and erythrocytic sharp separation.

Figure 70 is to use Paraformaldehyde 96 to be fixed on the cell enrichment assembly and a comparison film of the cell observed by fluorescent microscopy.Target cell combines with obstacle and capture component bottom.

Figure 71 A is that the cell counting of microfluidic separation of normal whole blood is with respect to the graphic representation of fluid cell dia.Figure 71 B comprises that circulating tumor cell group's the cell counting of microfluidic separation of whole blood is with respect to the graphic representation of fluid cell dia.Figure 71 C is the graphic representation of Figure 71 B, has shown that in addition the size of getting rid of most of natural hemocytes holds back.Figure 71 D is Figure 71 C₅Graphic representation, shown that in addition the cell mass of holding back greater than size can comprise endotheliocyte, endometrial cell or the trophoblast of pointing out morbid state.

Figure 72 relate to separate with the counting cells sample in maxicell and further analyze the synoptic diagram of the method for maxicell subgroup, wherein said counting prompting disease of patient state.

Figure 73 A is a kind of preferred determinacy Design of device of the present invention that is used for incorporating into.Figure 73 B is the form that relates to parameter corresponding to Figure 73 A.

Figure 74 be used for according to the present invention apparatus of the present invention magnetic separating device cross-sectional view strength and to be used for after the relevant treatment stream of cellular segregation be the release of off-line analysis.

Figure 75 is the assembling of magnetic separating device and functionalized synoptic diagram.Magnetized post makes it possible to carry out change after the packing of device.

The monoclonal antibody that Figure 76 is to use Transferrins,iron complexes (CD71) acceptor with magnetic separating device be applied to from composite mix for example blood catch and discharge the synoptic diagram of CD71+ cell.

The monoclonal antibody that Figure 77 is to use full Transferrins,iron complexes (CD71) acceptor with magnetic separating device be applied to from composite mix for example blood catch and discharge the synoptic diagram of CD71+ cell.The iron level of full Transferrins,iron complexes is abundant, commercially available acquisition, and than its similar monoclonal antibody have higher affinity constant and with the specificity of CD71 acceptor interaction.

Figure 78 is the synoptic diagram of high gradient magnet.Described magnet is designed to produce 1.2 teslas and～3 teslas/mm.

The synoptic diagram capillaceous of the configurations of magnets of the contiguous Figure 78 of Figure 79 A.Figure 79 B shows the graphic representation of the field intensity of magnet as the function of position in the kapillary.Figure 79 C is the erythrocytic picture that is concentrated into separated region in magnetic field after 10 minutes.

Figure 80 is from the precipitation (positive part) of the erythroblast of male Cord blood preparation and the picture of leukemic precipitation (negative part).At first use determinacy side tripping device to extract karyocyte, and use the 50M Sodium Nitrite to handle 10 minutes from blood.Make karyocyte pass through the magnetic post then, wherein erythroblast is retained.In post, magneticstrength is about 1 tesla, and field gradient is about 3000 teslas/m, and flow velocity is about 0.4mm/ second.The Dulbecco PBS damping fluid that use contains 1%BSA and 2mM EDTA is from post flushing white corpuscle and collect as negative part.Use identical damping fluid with the flow velocity wash-out erythroblast in 4mm/ second and collect as positive part.

Figure 81 is to use a series of fluorescence photos of the method for Figure 80 from the isolating erythroblast of maternal blood.Use fluorescence in situ hybridization (FISH) pair cell to dye.Use the water gauge note probe of α SAT-zone to identify X chromosome respectively, and use the redness in α SAT-zone and satellite III district and green colouring material to identify Y chromosome.Use DAPI (blueness) pair cell nuclear to redye.

Figure 82 shows that the method for using Figure 80 is from the maternal blood erythroblast in isolating different stage of maturity.Use Wright-Geimsa dyestuff pair cell to dye.

Figure 83 A and 83B have shown the result's of the method enrichment of using anti-CD71 antibody and Figure 80 (B) Photomicrograph.Sample among the A contain from 1mL blood＞200,000 karyocytes, and the every mL blood of the sample among the B contains 100-500 the karyocyte of having an appointment.A plurality of erythroblasts that the method for describing by Figure 80 obtains are above about 1000 times based on the enriching method of antibody.

Figure 84 has shown the synoptic diagram of three kinds of methods of the present invention.

Figure 85 shows the synoptic diagram of intact device of the present invention.

Figure 86 describes the nuclear isolating schema of fetal erythrocyte.

Figure 87 is the synoptic diagram curve of the lysis process of maternal blood sample.

Figure 88 be interested cell of enrichment and the enrichment of preferential cracking institute sample in the synoptic diagram of microfluidic methods of cell.At first, interested cell comes enriched sample, then by their interested cells of retention time selective splitting in cracked solution of control by being imported preferred passage based on size.

Figure 89 is from the synoptic diagram of non-interested non-cracked intact cell based on the nuclear microfluidic methods of apart cracked cells of interest.Non-interested cell is imported refuse, and nucleus is retained in the product stream of wanting.Figure 90 describes the schema that is used for isolation of fetal cells nuclear and the leukocytic optional method of parent.

Figure 91 uses constant clearance width and flow supply and the mobile synoptic diagram that extracts the apparatus of the present invention on border basically.

Figure 92 a is the synoptic diagram of manifold of the present invention.Figure 92 b is the photo of manifold of the present invention.

Figure 93 is the graphic representation as the percentage ratio of the viable cell of the function that is exposed to hypotonic cracked solution.

Figure 94 is the dissolved graphic representation as the whole blood of the function of time in the lysis buffer.

Figure 95 a describes the form that uses the nucleus recovery afterwards of the total lysis program of CarneyShi fixed solution as herein described.Figure 95 b shows a series of fluorescence micrographs that use the total cell pyrolysis liquid nucleus FISH sample result of the fixing mediation of CarneyShi.Nucleus is FISHed X (water), Y (green) and Y (redness) and uses DAPI to redye.

Figure 96 is a schema of describing cell and nucleus cracked each several part in detail.

Figure 97 is the synoptic diagram of the exemplary means of magnetic enrichment.

Figure 98 is the photo of the exemplary means of magnetic enrichment.

Figure 99 is to use the Photomicrograph of the erythroid cells of the inventive method acquisition.

Figure 100 is the monocytic Photomicrograph that has the RBC that includes in.

Figure 101 is to use the Photomicrograph of the trisomy 21 fetal cell of the inventive method enrichment.

The present invention describes in detail

The invention provides the analytical equipment and the method that are used for the enriched sample analyte.Usually, by the analyte in the sample or other components and magnetic field interaction generation enrichment.Analyte can be based on inherent magnetic (for example iron-protein), external magnetic (for example magnetic bead of bound analyte), or lack any inherence or external magnetic and enrichment.Enrichment can be based on the existing magnetic of sample component or based on the interaction of the reagent that can change (for example induce or add) magnetic and take place.Method and apparatus of the present invention can be used for producing the enriched sample of analyte, for example the red corpuscle fetal erythrocyte of maternal blood (for example from).

Magnetic separates

The inherent magnetic (for example changing in methods described herein) of analyte or external magnetic (for example providing by magnetic bead) can be used for separation, enrichment or the removing with respect to other components influence analytes of sample.Separation, enrichment or removing can comprise positive the selection, and the analyte of promptly wanting is attracted to magnetic field, maybe can use negative the selection, and the analyte of promptly wanting is not attracted to magnetic field, for example repulsion or unaffected.In arbitrary situation, the analyte group of containing required analyte can be collected and be used for analyzing or further handling.

Be used to implement the isolating device of magnetic and can be any device that can produce magnetic field, in one embodiment, MACS post (for example from Miltenyi Biotec) is used to influence the enrichment of magnetic response analyte.If analyte has magnetic response, for example by interacting with reagent as herein described, it can be attracted to the MACS post under magnetic field, thereby allows required analyte with respect to other components of sample and enrichment.In another embodiment, can use a kind of device to realize enrichment, described device is generally the microfluidic device that contains a plurality of magnetic obstacles.If the analyte in the sample has magnetic response (for example by combining with analyte with the reagent interaction of the inherent magnetic that changes analyte or by the particle with magnetic response), analyte can be in conjunction with obstacle, thereby allows the enrichment of bonded analyte.Alternatively, can use negative the selection.In this embodiment, the analyte that needs can not have maybe can be caused and not have magnetic response, or unwanted analyte can have or be caused and have magnetic response, or in conjunction with having the magnetic response particle.In this case, unwanted one or more analytes are retained in the magnetic devices, and the analyte of wanting is not retained in the magnetic devices, therefore with the enrichment of analytes wanted sample.

In another embodiment, before applying magnetic field, use the agent treated sample that comprises magnetic-particle.As described herein, use suitable catch part for example the combinative antibody of analyte wrap by magnetic-particle.The sample that magnetic field is put on processing is with the analyte of selectivity attraction in conjunction with magnetic-particle.

Passage or other zones of device can have magnetic response or can not have magnetic response.In one embodiment, analyte passage that passes through and the magnet coupling that can in passage, produce suitable magnetic field.Exemplary magnet is shown among Figure 78.Alternatively, the passage in the device contains the magnetic response zone, and it changes the magnetic field that is applied usually.Usually, magneticstrength is 0.05 to 5.0 tesla, for example about 0.5 tesla, and the magnetic response zone produces 100 to 1,000, the field gradient of 000 tesla/m, for example about 10⁴Tesla/m.

The magnetic area of device can be used hard or soft magnetic material is made, and described material is such as but not limited to iron, steel, nickel, cobalt, rare earth metal, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt-platinum and strontium ferrites.The part of device can directly assemble out from magneticsubstance, or magneticsubstance can be applicable to another kind of material.But the design of the use simplification device of retentive material, this is to need not other drivings because they can produce magnetic field.Yet soft magnetic material can only need by the material degaussing is discharged and downstream processing bonded analyte.According to magneticsubstance, application method can comprise the polymer-bonded thing of cathode sputtering, sintering, electrowinning or thin film coated-magnetic powder mixture.An embodiment preferred is by use polymer complex rotational casting thin film coated micromachine obstacle (for example silicon post), for example polyimide-strontium ferrous (polyimide is as tackiness agent, and strontium is ferrous as the magnetic weighting material).After applying, polymer magnetic coating is hardened to obtain stable mechanical properties.After sclerosis, will install and be exposed to the outside fast and induce induction field, it arranges the preferred orientations of permanent magnetic in device.The magnetic flux density in magnetic field and inherent coercivity can be controlled by magnetic weighting material volume percent.

In another embodiment, with electric conductivity material micro-structure in the outside surface of the microfluidic device that is wrapped up.This result can be made up of the single circuit with about 100 microns space periodicity.Layout by controlling this circuit and the current's intensity by this circuit can develop the period zones in the microfluidic device that higher and low magnetic intensity wrapped up.

In another embodiment, the magnetic response zone comprises the pearl that uses inviscid plastics or Teflon coating parcel.

For above-mentioned any embodiment, the magnetic field in any source can be used for the present invention, and can comprise hard magnetic body, soft magnetic bodies, electromagnet, superconductor magnet or its combination.In one embodiment, inhomogeneous permanent magnet in space or electromagnet can be used for producing magnetic-particle and the cyclic array (Deng etc., Applied Physics Letters, 78,1775 (2001)) produce magnetic-particle in some cases in structureless microfluidic channel of structure.Alternatively, can apply non-uniform magnetic field with regular periodicity.Can use electromagnet in device, to produce uneven magnetic field.Non-uniform magnetic field produces higher and than the zone of low magnetic field intensity, and they attract magnetic-particle in the periodic arrangement mode in device.Other external magnetic fields can be used for producing magnetic-particle bonded magnetic regions.Retentive material yet is used for the assembling of device, thereby has got rid of the needs to electromagnet or external magnetic field, and in one embodiment, described device contains a plurality of passages with magnetic regions, for example increases the volume treatment capacity.In addition, these passages can vertically superpose.

In the above-described embodiment, can discharge from the restriction site in the passage, for example flow through the fluidic overall flow rate of device, reduce magnetic field by increase in conjunction with the analyte of magnet, or by the two certain combination.

Magnetic field can be adjusted to influence paramagnetic particle above-mentioned and that have the magnetic mass susceptibility, for example 0.1-200 * 10^-6m³/ kg scope.Employed paramagnetic particle can be based on size classes: particulate (1-5 μ m cell dia size); Micella (approximately 100nm); And (the approximately 2-10nm) of molecule.The fundamental force that acts on the paramagnetic entity is:

$F_b=\frac{1}{{2\mathrm{\&mu;}}_0}\mathrm{\&Delta;\&chi;}{V}_G\&dtri;{\mathrm{<figure-callout\; label="B"\; filenames="A20068004166601281.png,A20068004166601461.png"\; state="\{\{state\}\}">B</figure-callout>}}^2$

Work as F_bBe to act on to have V_bDuring the magnetic force of volumetrical paramagnetic entity, Δ χ is the difference of the magnetic susceptibility between magnetic-particle χ b and the surrounding medium χ f, μ₀The magnetic that is freeboard is permanent, and B is the external magnetic field,Be that gradient is handled factor.But magnetic field Be Controlled and adjusting make it possible to attract and keep particulate, micella and the molecule paramagnetic entity of wide region.

Can change the reagent of magnetic

In certain embodiments, other components of analyte or sample and the inherence that can change analyte or other components or the reagent react of external magnetic.Exemplary agents comprises the reagent of oxidation or reduction transition metal, magnetic bead that can bound analyte, or can chelating or in conjunction with the reagent (for example as U.S. Patent No. 4,508,625 descriptions) of iron, or other magneticsubstances or particle.Concrete reagent comprises chemical reagent, Sodium Nitrite for example, and gas is nitrogen for example, oxygen, carbonic acid gas, carbon monoxide or its mixture.For example, but reagent paramagnetic or degaussing analyte.Reagent also can make the analyte deoxidation, for example myohaemoglobin or oxyphorase.Reagent can change the magnetic of analyte, makes it can reduce or increase its magnetism to magnetic field, can reduce or increase its repulsive force to magnetic field, or elimination magnetic makes analyte not affected by magnetic fields.Reagent also can change wherein the fluidic magnetic of dissolving, suspending or carrying analyte, or the magnetic of cell cytoplasm.Reagent also can change the rheological of analyte.

In certain embodiments, magnetic-particle combines with analyte and gives external magnetic response.For these embodiments, any particle that reacts with magnetic field can be applicable to apparatus and method of the present invention.The ideal particle is that have can be by those of the surface chemistry of chemistry or physical modification, for example by chemical reaction, physical adsorption, coiling or electrostatic interaction.Magnetic-particle of the present invention can be virtually any size and/or shape.In certain embodiments, magnetic-particle has the diameter less than 500nm, 400nm, 300nm, 200nm, 100nm, 90nm, 80nm, 70nm, 60nm or 50nm.In certain embodiments, magnetic-particle has the diameter of 10-1000nm, 20-800nm, 30-600nm, 40-400nm or 50-200nm.In certain embodiments, magnetic-particle has the diameter greater than 10nm, 50nm, 100nm, 200nm, 500nm, 1000nm or 5000nm.Magnetic-particle can be exsiccant or liquid form.Fluid sample can take place by any known method in this area with mixing of second liquid medium that contains magnetic-particle.

Catching part can combine with magnetic-particle by any known method in this area.The example comprises chemical reaction, physical adsorption, coiling or electrostatic interaction.Partly depend on the shape of the analyte of target in conjunction with catching of magnetic-particle.The example of catching part includes but not limited to protein (for example antibody, avidin and cell surface receptor), electrically charged or uncharged polymkeric substance (for example polypeptide, nucleic acid and synthetic polymkeric substance), hydrophobic or hydrophilic polymer, small molecules (biological example element, receptors ligand and sequestrant) and iron.The part of catching like this can be used for specificity in conjunction with cell (for example bacterial cell, pathogenicity bo cell, fetal cell, fetal blood cell, cancer cells, epidermic cell, endotheliocyte and hemocyte), organoid (for example nucleus), virus, peptide, protein, polymkeric substance, nucleic acid, supramolecular complex, other biological molecule (for example organic or inorganic molecule), small molecules, iron or their combination or fragment.The specific examples of catching part comprises anti-CD71, anti-CD36, anti-GPA, anti-EpCAM, anti-E-cadherin, anti-Muc-1 and full Transferrins,iron complexes.Other concrete antibody are described at this paper.In another embodiment, catching part is fetal cell (for example fetal erythrocyte), cancer cells or epidermic cell.

Sample also can combine with the reagent of the inherent magnetic that changes analyte.The analyte that changes can be caused by reagent with respect to unaltered analyte to have more or less magnetic response and maybe can cause and do not have magnetic response.In one embodiment, use Sodium Nitrite to handle and contain sample (for example, being eliminated the erythrocytic maternal blood sample of parent) the maternal blood sample of fetal erythrocyte (fRBCs), thereby cause the oxidation of the foetal haemoglobin that contains among the fRBCs.This oxidation changes the magnetic response with respect to the foetal haemoglobin of other components of sample, parent leukemia for example, thus allow to separate fRBCs.In addition, the otherness oxidation of fetus and mother cell can be used for isolating fetal and parent has nuclear RBCs.Be present in oxyphorase (for example adult or fetus), myohaemoglobin or the cytopigment (for example cytochrome C) contain the magnetic response component for example any cell of iron can be modified to and change for example inherent magnetic response of cell or its component (for example organoid) of analyte.

In addition, cell can with induce, prevent, increase or reduce to have the protein of magnetic response or the reagent of other developed by molecule contacts.For example, the carrier of coding magnetic susceptible protein matter or subunit's (for example oxyphorase or heme) is with interested initial gene transfection.Then, successful cells transfected can be used magnetic device enrichment as herein described.Such method allows the target of sharp separation transient transfection before loss of function or allows to set up the group of stable transfection.Transform that magnetic susceptible protein matter is expressed so that it depends on envrionment conditions or existence or does not have activator is possible.Such preparation also can be used for inducing the intracellular expression of the magnetic susceptible protein matter with natural but dormancy.

The enrichment based on magnetic of various modes

Method of the present invention also can be used for enrichment of cell or other particles simultaneously based on inherent or external magnetic.For example, in a blood sample, can use inherent magnetic resolution red corpuscle, and use in conjunction with for example after CD45 or the leukocytic magnetic bead processing of CD 15 antibody, handling white corpuscle simultaneously.Such multimodal method allows non-magnetic response sexual cell or component from example enrichment.For example, the stem cell of Cord blood can separate with white corpuscle from red corpuscle.

Analytical equipment

Device of the present invention can be united any analytical equipment application or be comprised any analytical equipment.The example comprises affinity column, cell counter, grain sorting instrument, for example fluorescence activated cell analyser and magnetic activating cells analyser, capillary electrophoresis, sample storing unit, and sample preparation apparatus.Microfluidic device is for being interested especially with system described herein bonded.

The exemplary analysis device comprises and can be used for the separation of particulate based on size, shape or deformability, comprising: strainer, screen cloth and determinacy tripping device, for example international publication No.2004/029221 and 2004/113877, Science such as Huang 304,987-990 (2004), the U.S. announces No.2004/0144651, U.S. Patent No. 5,837,115 and 6,692,952 and U. S. application No.11/449,161,11/227, those that describe in 904 and 11/449,149; The device that can be used for affinity capture, for example country announces that the No.2004/029221 and the U.S. announce those that No.2005/0266433 describes; The preferential cracked device that can be used for cells in sample, for example international publication No.2004/029221, U.S. Patent No. 5,641,628 and the U.S. announce No.11/449, those of 149 descriptions; Can be used for disposing the device of cell, for example international publication No.2004/029221, U.S. Patent No. 6,692,952 and the U.S. announce those that No.2004/0166555 and 2006/0128006 describes.Two or more identical or different devices can unite in proper order or incorporate in the single assembly, for example describe among the international publication No.2004/029221.

In specific embodiments, analytical equipment can be used for different analytes in the enriched sample, for example is used for collecting or further analyzing.Rare cells or its component are compared and can be retained in the device with other cells of (for example describing among the international publication No.2004/029221) as described, or by enrichment.Per sample, exemplary rareness comprises fetal cell (for example fetal erythrocyte), stem cell (for example undifferentiated), cancer cells, immune system cell (host or graft), epidermic cell, phoirocyte, bacterium, fungi, virus and pathogenic agent (for example bacterium or protozoon).Can be from the such rare cells of the sample separation that comprises body fluid, for example blood or environment are originated, and for example water or air sample can be from the maternal peripheral blood liquid enrichments.Fetal erythrocyte for example is used for determining the cell of developmental fetus and identifies dysploidy or hereditary property, for example sudden change.Can be used at making progress from peripheral blood enrichment cancer cells with monitor therapy.Also can be to body fluid or environmental sample screening pathogenic agent, for example septicemia or bacterium or viral meningitis of intestinal bacteria, blood borne disease for example.Rare cells also comprises the cell from a kind of organism that exists in another organism, for example from the cell of transplant organ.The analyte of reservation or enrichment can for example use for example fluorescence or radioactive probe mark in the device, stands chemistry or genetic analysis (for example fluorescence in situ hybridization), if biological, then can cultivate or observe or use probe in detecting.Analytical equipment can comprise or not comprise microfluidic channel, promptly can be or can not be microfluidic device.The two-dirnentional structure of the passage of the device of importing analyte can be depending on the size or the type of the analyte of application.Preferably, the passage in the analytical equipment has and is not more than 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 at least one dimension (for example height, width, length or radius) of 1mm.The microfluidic device of using in the system and method described herein preferably has less than 1,0.9,0.8,0.7,0.6,0.5,0.4,0.3,0.2,0.1 or even at least one dimension of 0.05mm.Those skilled in the art can determine the preferred dimension of analytical equipment based on required purposes.

Analytical equipment (for example determinacy device) but coupling or comprise the bank that contains the reagent (magnetic-particle that for example has bound fraction or Sodium Nitrite) that can change analyte (for example cell, for example red corpuscle) magnetic.This bank can or can form integral body with it from the analytical equipment separation.Can comprise by any method that diffusion, mechanically mixing, unordered mixing, convection current or eddy current carry out mixing of reagent and analyte.Reagent can be in bank stored dry and when introducing sample, liquefying, or be stored in the solution and and sample mix.In another embodiment, in the transmission sample, continuously or with the high density of separating reagent is added into bank.

Described bank also can comprise the analyte of permission change and the separation of by-products of unreacted reagent or reagent and analyte response.For example, as described herein, determinacy is separated and be can be used for this purpose.Alternatively, can use strainer, flushing or additive method.The part that such device can be used as bank or analytical equipment is comprised or is not comprised as the part of bank or analytical equipment.

In one embodiment, in the textured surface that the analyte by passage can contact, described bank has the magnetic region, for example by the zone in magnetic-particle and the passage is adhered to.With respect to the dimension of passage,, can provide the interactional structure that strengthens between analyte and the bonded magnetic-particle by for example suitable selection of magnetic-particle size and dimension of parameter.Can use can be by the suitable for example antibody (for example anti-CD71, anti-CD36, anti-CD45, anti-GPA, antigen i, anti-CD34, anti-foetal haemoglobin, anti-EpCAM, anti-E cadherin or anti-Muc-1) of part of catching of affine machine-processed bound analyte.Magnetic-particle can be uniformly distributed in whole device or space analysis district.In addition, magnetic-particle is used in and produces structure in the device.For example, can be used for attracting magnetic-particle and form " bridge " that connects two zones in two magnetic area of passage opposite side.But the magnetic-particle magnetic adherence is in the hard magnetic district of passage or be attached to the soft magnetism district that is actuated to produce magnetic field.

An example of bank is shown in Figure 74, and it has been described to separate and discharge target analyte then and has for example flowed from the cell of compounding mixture or the bank geometrical shape and the function treatment of molecule.As shown, this bank contains the obstacle that extends to relative channel surface from a channel surface.Obstacle can extend or not extend whole distance through passage.In this embodiment, obstacle has magnetic (for example containing the position, upfield in hard or soft magnetic particles or the non-uniform magnetic-field) and attracts and the reservation magnetic-particle, and it can use and catch the part bag can be the cell that is attracted to magnetic field maybe.Can change the geometrical shape of bank, the distribution of obstacle, shape, size and flow parameter are for example caught the analyte of part (for example as describing among the international publication No.2004/029221) attraction in conjunction with magnetic-particle by use to optimize the bioaccumulation efficiency of interested analyte.In a specific embodiment, the silicon wafer that distinct configuration (through equilateral triangle, cornerwise interval and density and random arranged distribution) uses and to have different shapes the microstructure magnetic obstacle of (cylindrical, rectangle, trapezoidal or multiform) and size (10-999 micron) to carry out anodal covering, with the collision frequency of maximization change or unaltered analyte, obstacle is in the boundary line of continuous irrigation beam.That the definite geometrical shape of magnetic obstacle and the distribution of obstacle can be dependent on is separated, the type of the analyte of enrichment or purifying.

Figure 75 has described bank assembling and functionalized embodiment.Magnetized obstacle makes it possible to carry out modify after the packing of bank.Semiconductor process parameters (the solvent edge sealing agent of high heat, fixed lid) makes this device have ubiquity with the uncompatibility of catching part (to temperature sensitive and inorganic and organic solvent) and is suitable for using all to catch part carries out functionalized.By using magnetic field to catch the reservation of part on obstacle (for example post) is to be better than using the composite surface chemistry to carry out the advantage of fixed prior art.Bank can make the easy and optional fast mixture of catching partly or catching part that uses of terminal user load bank, thereby increases the diversity of using.By this bank can make it possible to as required and a step of " on time " functionalized, thereby if catch part when producing by chemically crosslinked then walk around the stability of the validity period of catching part.Can load and remain in cluster of differentiation (CD) acceptor that partly includes but not limited on the mammalian cell of catching on the obstacle, synthetic and the reorganization part of cell receptor, and any other organic and inorganic molecule, maybe can be attached to the compound of interest of any magnetic-particle.

Other assemblies

Device of the present invention also can comprise and be used for for example separating, the element of collection, operation or check and analysis thing.Such element is known in the art.For example, device of the present invention (device of for example incorporating the determinacy device into also can comprise be used for broad variety separate comprise affine, based on the isolating assembly of cracked separation, electrophoretic separation, centrifugation and two dimensional electrophoresis.Device of the present invention also comprises the assembly that is used for from the two-dimensional imaging of device output, for example microwell array or plane surface.Such microwell array or plane surface can pass through microscope or for example Kamera imaging or the observation of other vision instruments.

Device of the present invention also can be united other enriching apparatus application, at same apparatus or different device.Other beneficiation technologies are in for example international publication No.2004/029221 and 2004/113877, U.S. Patent No. 6,692,952, the U.S. announces No.2005/0282293 and 2005/0266433 and U. S. application No.11/449, describes in 161, and they incorporate the application separately by reference into.

Determinacy is separated

In one embodiment, the invention provides a kind of device, it comprises that determinacy imports particulate passage and the magnet based on the hydromeehanics size, for example unites the bank that contains the reagent that can change particulate magnetic.The present invention also provides a kind of method that is used for producing with first analyte with respect to second analyte sample of enrichment, described method by with sample application in comprising based on hydromeehanics size determinacy deflection particulate passage, thereby second sample of first analyte that produced enrichment, the reagent of uniting described second sample and the change first analyte magnetic, or depend on the magnetic of existence, thereby and apply magnetic field and separate first analyte and second analyte.

In one embodiment, passage comprises the one or more obstacle arrays that allow fluid components determinacy sidesway.Such device is at for example Huang etc., and Science 304,987-990 (2004) and the U.S. announce among No.2004/0144651 and the international publication No.2004/029221 and describes.These devices can further be used the gap network array, the fluid that wherein flows through the gap by be divided into unequally continue after the gap.In one embodiment, even the gap equates that on dimension the fluid that flows through the gap is also separated unequally.Fluid carries particle to be separated by the gap array.Sight line according to array is arranged mobile with little angle (flow angle).Have particle greater than the hydromeehanics size of critical size along the migration of the sight line in the array, follow mobile with different directions less than those of the hydromeehanics size of critical size and have.Being flowing under the laminar flow condition in the device takes place.

Critical size is the function of several design variables.Mention the obstacle array among Fig. 1, every row's obstacle moves Δ λ with respect to previous row of horizontal, and wherein λ is the distance (Figure 1A) of the center to center between the obstacle.(bifurcation ratio, ε) decision branches to the mobile ratio in the left side of next obstacle to parameter Δ λ/λ.In Fig. 1, describe for convenient, ε is 1/3.Usually, if the stream between two obstacles is Φ, inferior stream is ε Φ, main flow be (1-ε Φ) (Fig. 2).In this embodiment, the stream by the gap is divided into three (Figure 1B) basically.Although three tributaries by the gap become waveform to flow around obstacle, the mean direction in each tributary is at the mobile general direction.Fig. 1 C has described analyte the moving by array of size greater than critical size (for example 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 analyte moves with main flow, is extremely passed through the main flow in each gap by sequential transfer.

With reference to figure 2, critical size is about 2R_Critical, R wherein_CriticalBe the streamline of stagnation and the distance between the obstacle.As fruit granule for example the barycenter of cell drop on R_CriticalOutside, then particle is followed main flow and is moved along the sight line of array.If known flow pattern through the gap can be determined R_Critical(Fig. 3).R_CriticalBe to form time thickness of the stream layer of stream.For given gap size d, can shear R based on bifurcation ratio_CriticalUsually, ε is more little, R_CriticalMore little.

In the array of determinacy sidesway, particulate behavior of different nature has different size (Fig. 4) as them.For example, lymphocyte is to have～5 μ m diameter sphere, and red corpuscle is to have～the bi-concave body of 7 μ m diameters and～1.5 μ m thickness.Erythrocytic major axis (diameter) is greater than lymphocytic diameter, but its minor axis (thickness) is less.If when the red corpuscle arrangement along them to the mobile major axis when the obstacle array drives of being flowed, then their size is their thickness (1.5 μ m) effectively, and is littler than lymphocyte.When red corpuscle by fluid flow when the obstacle array drives, it tend to arrange along it to the mobile major axis and its behavior similar～the wide particle of 1.5 μ m, effectively " less than " lymphocyte.Therefore, although the volume of analyte can separate them according to the shape of analyte by identical described method and apparatus.In addition, the behavior with analyte of different deformabilities has different size (Fig. 5) as them.For example, two kinds of analytes with identical not deformed shape can separate by the determinacy sidesway, and this is because when the obstacle in a kind of analyte contact array wherein and when changing shape, comparable another kind of analyte is easy deformation more.Therefore, can be based on the separation in any parameter implement device that influences physical dimension, shape and deformability that the hydromeehanics size comprises analyte.

With reference to figure 6, meaning has the mixture of the analyte of different fluid mechanics size such as cell and can produce two kinds of products at bottom collection analysis thing from array top covalency as shown, contains greater than critical size 2 R_CriticalThe output of analyte, contain output less than the cell of critical size.When for example sample classification being two or more inferior samples, can collect arbitrary output or output the two.Analyte greater than gap size obtains to catch in array.Therefore, array has the working size scope.Cell must be greater than holding back size (2 R_Critical) and less than maximum by size (array gap size) to be imported into main flow." size range " of array is defined as maximum by size and the ratio of holding back size.

Separation from the free unreacting reagent of the analyte that changes

Can use the determinacy device to separate free unreacting reagent from the analyte that changes.Shown in Figure 60, can at labelled reagent such as antibody be introduced into the determinacy device or before the determinacy device, but operational analysis thing (for example cell sample) preincubation.Ideally, reagent specifically with interested analyte response, for example cell mass, for example epidermic cell.Exemplary indicia reagent comprises antibody, quantum dot (quantum dots), phage, fit, the molecule that contains fluorophor, can carry out detecting the enzyme of chemical reaction, changes the reagent (for example Sodium Nitrite) or the functionalized pearl of magnetic.Usually, the more interested analyte of reagent (for example cell) or little in conjunction with the interested analyte of pearl, when being introduced into device with reagent bonded sample, unreacted reagent moves along undeflected device, and the analyte (for example analyte of binding reagents) that changes is deflected, thereby separates the analyte of unreacted reagent and change.Advantageously, this method has realized separating of apart and free unreacting reagent and analyte.In addition, if desired, this separation method helps the downstream sample analysis, and does not need release steps or potential destructive method of analysis, and is as described below.

Figure 61 has shown a kind of Special Circumstances, and wherein, the mark sample of enrichment contains because of having similar size and target cell isolating non-target cell group altogether.Non-target cell does not disturb the downstream sample analysis of the detection that depends on bonding mark reagent, and this is in conjunction with interested cell because of this reagent selectivity.

The array design

Can use the obstacle realization determinacy in gap array and the passage to separate.The exemplary configuration on such array, by-pass channel and border is as described below.

The unistage type array.In one embodiment, single hop contains the obstacle array, for example cylindrical pillars (Fig. 1 D).In certain embodiments, for example when separating white corpuscle and red corpuscle, array has greater than the largest passages size of holding back several times of sizes.This structure can be used the associating of big gap size and little bifurcation ratio ε and obtain.In preferred embodiments, ε is 1/2 at the most, for example at the most 1/3,1/10,1/30,1/100,1/300 or 1/1000.In such embodiments, the obstacle shape can influence the flow pattern in the gap, yet obstacle can be compressed in flow direction, so that array shortens (Fig. 1 E).The unistage type array can comprise by-pass channel as herein described.

The multi-stage type array.In another embodiment, the multi-stage type array is used to separate the analyte of wide size range.An exemplary means as shown in Figure 7.Shown device has three sections, has any amount of section but can use, and array can have many sections as required.Usually, hold back size greater than the size of holding back in second section in first section, and first section the maximum of holding back second section of dimensional effect is by size (Fig. 8).For after the section also be like this.Before the analyte that for example causes in second section stopping up arrives second section first section deflection (and removal) they.Similarly, before the analyte that causes in second section stopping up arrives the 3rd section second section deflection (and removal) they.

As described, in the multi-stage type array, for example at first deflection of cell quilt of big analyte that can cause the downstream to be stopped up, these analytes that are deflected need be walked around tract to avoid obstruction.Therefore, device of the present invention can comprise the by-pass channel of removing output from array.Although described the analyte of removing greater than critical size here, by-pass channel also can be used for removing output from any part of array.

The different designs of by-pass channel is as follows.

Single by-pass channel.In this design, all sections have a by-pass channel, or only have one section.The physical boundary of by-pass channel limits (Fig. 9-11) by array boundary with at opposite side by sidewall in a side.Single by-pass channel also can be used (Figure 12) with the diad array.

Single by-pass channel also can be designed to be connected to keep the steady flow (Figure 13) by device with array.As shown, by-pass channel has the different in width that is designed to keep by all sections steady flow, makes flowing in the passage not disturb flowing in the array.Such design also can be used (Figure 14) with the array bigeminy.Single by-pass channel also can be designed to be connected to keep the substantially invariable fluid resistance (Figure 15) by all sections with array.Such design also can be used (Figure 16) with the array diad.

Many by-pass channels.In this design (Figure 17), each section has the by-pass channel of himself, and passage separates by sidewall mutually.Big analyte for example cell is deflected in the main flow and is deflected to (by-pass channel 1 among Figure 17) in the by-pass channel to first section lower right corner then.Do not cause second section obstruction continue to second section than minicell, be deflected to second section the lower right corner and another by-pass channel (by-pass channel 2 among Figure 17) unexpectedly greater than the cell of second section critical size.Can repeat this design according to many sections needs.In this embodiment, by-pass channel not fluid connects, and allows collection or other operations of a plurality of parts.By-pass channel needs not be straight or physics parallel (Figure 18) each other.A plurality of by-pass channels also can be used (Figure 19) with the diad array.

A plurality of by-pass channels can be designed to be connected to keep the steady flow (Figure 20) by device with array.In this embodiment, by-pass channel is designed to remove flow and makes flow not multilated, i.e. substantially constant in the array.Such design can also be used (Figure 21) with the array diad.In this design, can share the by-pass channel of central authorities between two arrays in the diad.

The optimal boundary design.If the array infinity, the flow distribution of each gap location is identical.Stream Φ through the gap is identical, and time stream in each gap is ε Φ.In the practice, no current-limiting type is upset on the border of array.The part on the border of array can be designed to produce the flow pattern of unlimited array.The border can be to flow to supply with, and promptly array is injected with fluid in the border, or the extraction of flowing, promptly fluid is extracted from array in the border.

Preferred flow extract the border gradually broadening to extract ε Φ (arrow is represented Figure 22) from each gap at boundary (d=24 μ m, ε=1/60).For example, the distance between array and the sidewall increases gradually to allow adding ε Φ from each gap to the border.Because this border design, the flow pattern in this array is not subjected to the influence of by-pass channel.

The preferred supply boundary that flows narrows down gradually accurately to supply with ε Φ (arrow is represented among Figure 23) at boundary (d=24 μ m, ε=1/60) to each gap.For example, the distance between array and the sidewall reduces gradually to allow removing ε Φ from the border to each gap.Once more, because this border design, the flow pattern in this array is not subjected to the influence of by-pass channel.

Mobile supply boundary is also can be the same with the gap of array wide or than the latter wide (Figure 24) (d-24 μ m, ε=1/60).If for example to allow the collection analysis thing, broadside circle suits the requirements as by-pass channel on the border.Can use and use its border (in Figure 24 arrow represent) of complete mobile part to supply with array and ε Φ to be supplied to each gap on the border.

Figure 25 has shown the single by-pass channel (ε=1/10, d=8 μ m) in the diad array.By-pass channel comprises two mobile supply boundaries.The stream of dottedline 1 is the Φ bypass in by-pass channel---Φ stream adds from the gap to the Φ bypass in dotted line left side.The shape of adjusting the boundary obstacle makes that each gap location that is flowing in the border that enters array is ε Φ.The stream atdotted line 2 places is again the Φ bypass.

Resistance to flow device on the wafer of restriction and steady flow

But determinacy is separated also applicating fluid resistance gauge with flowing and also limit from flowing that array is collected in restriction and the stable array.Figure 26 has shown the synoptic diagram of plane device, and sample for example blood, access road, damping fluid access road, Waste outlet passage and product exit passageway is connected with array separately.Entrance and exit works as the resistance to flow device.Figure 26 has also shown the corresponding fluids resistance of these different device assemblies.

Flow limitation in the array

Figure 27 and 28 has shown the electric current and the respective width of sample, and damping fluid flows in array when device has constant depth and operates with the setting pressure difference.Determine to flow by the pressure difference that resistance separates.In this special device, I_BloodAnd I_BufferIdentical, and this has determined the same widths of blood and damping fluid stream in the array.

Collect the restriction of part

By the comparative resistance and the Waste outlet passage of control product, collection tolerance that can the condition each several part.For example, in this special picture group, work as R_ProductGreater than R_RefuseThe time, it is cost and waste part dilution that denseer product part will cause with potential increase loss.Otherwise, work as R_ProductLess than R_RefuseThe time, product rarer and more high yield part will pollute by waste streams be that cost is collected with potential.

Stability of flow

Each entrance and exit passage can be designed such that the pressure difference through passage is suitable for or drive the fluctuation of pressing greater than total.In normal conditions, the entrance and exit pressure difference is for driving 0.001 to 0.99 times that presses.

Multichannel determinacy array

Can use multichannel determinacy array to realize the determinacy separation.Multichannel array is placed the treatment capacity that increases sample preparation on the device, and allow parallel processing different piece or a plurality of samples of operation or the part of sample.Frequency multiplexing technique also is that preparatory application is required.The simplest multiple-pass unit comprises that order is two devices that cascade connects.For example from the output terminal of the main flow of a device can with the input terminus coupling of the twoth device.Alternatively, a device time stream output terminal can with second the device the input terminus coupling.

Bigeminy.Two arrays can dispose side by side, for example mirror image (Figure 29).In such configuration, the critical size of two arrays can be identical or different.In addition, array configurations can be become to make the border of main current flow to two array, the edge that flow to each array or their combination.Such multichannel array also can contain and is configured in central area between the array, for example to collect greater than the analyte of critical size or to change sample, for example by buffer exchange, reaction or mark.

Frequency multiplexing technique on the device.Handle forming diad, can on same apparatus, dispose two or more arrays (Figure 30 A) with inlet separately.Such configuration can be applicable to a plurality of samples, or a plurality of array can be used for the identical sample of parallel processing with identical inlet connection.In the parallel processing same sample, outlet can be or can not be that fluid connects.For example, when a plurality of arrays had identical critical size, outlet can be connected to be used for the high-throughput sample preparation.In another embodiment, array can not be all to have identical critical size or the analyte in the array can not be all to handle in the same manner, and outlet can not be that fluid connects.

Also can realize multichannel (Figure 30 B) on the single assembly by a plurality of diad arrays are placed.A plurality of diads or single array can be placed with any possible three-dimensional relationship each other.

Exemplary multi-stage type device.Except above-mentioned those, following exemplary multi-stage type determinacy device also can be included in the device of the present invention.For example, Figure 58 A has shown " cascade " configuration, and theoutlet 1 of one of them device is bonded to the sample inlet of second device.This allow to use the initial process step of first device, has made to the example enrichment of having introduced second device interested cell.According to the purpose purposes, two devices can have identical or different critical size.

In Figure 60, unlabelled cell sample is introduced in first device through the sample inlet cascade, and the damping fluid that will contain labelled reagent is introduced first device through fluid intake.Epidermic cell is deflected and the central outlets from the damping fluid that contains labelled reagent occurs.Then, the mark sample of this enrichment is introduced second device through the sample inlet cascade, and damping fluid is added in second device through fluid intake.Realize further enrichment of target cell and separating of free label reagent, and can further analyze the sample of enrichment.Alternatively, can before being introduced second device, labelled reagent labelled reagent be added directly to the sample that occurs from the central outlets of first device.The use of cascade configuration can allow to use in a small amount or the labelled reagent of higher concentration with the cost that the single device less than Figure 60 disposes, and in addition, uses first existence of installing by initial process step significantly to reduce generable any non-specific binding.

But an arrangement of two or more device sections is " band is logical " configurations.Figure 58 B has shown this configuration, and theoutlet 2 of one of them device is bonded to the sample inlet of second device.This allows to use the initial process step of first device, makes the sample of introducing second device contain and is retained in the cell that is not deflected in first device.When interested cell was not cell maximum in the sample, this method was useful, and in this case, first section can be used for being deflected to central outlets and removing them by permitting great non-target cell.As in cascade configuration, according to the purpose purposes, two devices can have identical or different critical size.For example, with respect to the purposes that needs separate less endotheliocyte, different critical size needing to be suitable for the purposes of separating table chrotoplast.

In Figure 66, the cell sample of applying marking reagent preincubation is introduced the sample inlet of first device of bypass configuration, and through fluid intake damping fluid is introduced first and install.Dispose first device in the following manner: promptly big non-target cell is deflected and occurs from central outlets, and the mixture of target cell, little non-target cell and labelled reagent occurs from theoutlet 2 of first device.Then, this mixture is introduced second device through sample inlet, and damping fluid is added into second device through fluid intake.Realize the enrichment of target cell and the classification of free label reagent, and can further analyze the sample of enrichment.In the method, the non-specific binding of the cell of the deflection in labelled reagent and first section is an acceptable, and this is because cell and any bonded labelled reagent of deflection are removed from system.

In above-mentioned any many determinacy device configuration, the tie-in module that installs and be bonded to them can be integrated in the single assembly.For example, comprise that the single cascade unit of two or more sections is possible, as comprise the single shunting device of two or change section.Then with the input terminus coupling of the output terminal and the bank of multistage.

Little blot array.The determinacy device can also have the characteristics of little trace.The trace of removing array can reduce cost, and the quantity that reduces to collide with obstacle is to eliminate any potential physical disturbance or other influences to analyte.If the border between the section is not vertical with the length of mobile direction multi-stage type array, can reduce the length of multi-stage type array.Because the quantity of section increases, it is important that length reduces to become.Figure 31 has shown little trace syllogic array.

The purposes of apparatus of the present invention

As described, the present invention relates to be used for enrichment of analyte for example particle comprise the apparatus and method of bacterium, virus, fungi, cell, cellular component, virus, nucleic acid, protein and protein complex.The example of the fluid sample that the present invention considers comprises biologicfluid sample, for example the secretory product and the amniotic fluid of whole blood, sweat, tear, ear effluent, sputum, lymph liquid, marrow suspension, lymph, urine, saliva, seminal fluid, vagina effluent, cerebrospinal fluid, brain fluid, ascites, breast, respiratory tract, enteron aisle and genitourinary tract.In addition, system and method for the present invention has also been considered any other biological sample (for example biopsy samples) of solubilized or suspension.Except enrichment, device also can be used for the analyte in the sample is implemented different operating.Such operation comprises and changes for example magnetic or carry the fluid of analyte of analyte self.Preferably, device is used for from heterogeneous mixture enriching rare analyte, or changes rare analyte, for example by the liquid in the replacing sample or by analyte is contacted with reagent.Such device allow with to the analyte of potential fragility for example cell have limited discharge head enrichment, wherein the device of this aspect provides in the cell mechanical lysis that reduces or the cell and has activated.

Although mainly described the cell aspect, device of the present invention can be with can for example being used based on the enrichment of hydromeehanics size based on any analyte and/or other enriching methods as herein described of magnetic (or it lacks) separation, enrichment or removing.

Determinacy device and other analytical equipments can be used for concentrating sample, and for example wherein analyte is in contact with one another, water power interacts, or the flow distribution around the another kind of analyte is exerted one's influence.For example, the determinacy device can separate white corpuscle and the red corpuscle from the whole blood kind of human donor.Human blood contains by volume usually～45% cell.When stream of cells is crossed array, their mutual physics contact and/or couplings.The graphic cell physics contact mutually that has shown dense filling in the array of Figure 32.

As described, apparatus and method of the present invention can relate to inherent or one or more analytes of external magnetic resolution based on one or more analytes.In one embodiment, use the agent treated sample that changes analyte magnetic.Described change can maybe can be passed through the reagent mediation of the built-in magnetic of change analyte by the magnetic-particle mediation.Then, the magnetic response analyte can be attracted to the surface of device, and the required analyte in the sample (for example rare cells, for example fetal cell, pathogen cells, cancer cells or bacterial cell) can be retained in the device.In another embodiment, required analyte is retained in the device by the mechanism based on size, shape or deformability.In another embodiment, use negative the selection, wherein unwanted magnetic susceptibility analyte is combined in device, and required analyte is not combined.Except combination, the path of magnetic susceptibility analyte can be changed by magnetic field, for example required or unwanted analyte is imported specific direction, for example to outlet.Any embodiment can use the MACS post to be used for the retention analysis thing analyte of magnetic-particle (for example in conjunction with).

In using positive embodiment of the present invention of selecting, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% target analyte is retained in that (for example magnetic is bonded to device) is desirable in the sample of enrichment.The surface of device is designed to minimize the non-specific binding of non-target analyte ideally.In addition, at least 99%, 98%, 95%, 90%, 80% or 70% non-target analyte preferably is not retained in the sample of enrichment, for example magnetic joining device not.Selective retention in the device can cause specific analyte group, and for example blood, sputum, urine and soil, air or water sample separate from mixture.

Method and apparatus of the present invention allows to produce has highly purified enriched sample, for example wherein at least 0.01%, 0.1%, 1%, 10%, 20%, 50%, 60%, 70%, 80%, 90% or even 95% enriched sample be required analyte.When analyte is a cell for example when fetal erythrocyte or epidermic cell, high purity caters to the need especially, and this is because it allows to be used for quantifying PCR method.Device of the present invention also allows to have the high purity (for example keeping required analyte) of high yield.For example, the required analyte that is present at least 90% in the sample for example fetal erythrocyte is retained in the sample of apparatus of the present invention enrichment, and at least 90% for example at least 95% or even 99.9% unwanted analyte for example white corpuscle be not retained in the sample of enrichment.

In other embodiments, device of the present invention can be used for separating and checks blood-borne pathogens, bacterium and virus load, dissolves or be suspended in airborne pathogenic agent in the water medium, the pathogen detection that is used for foodstuffs industry, and the sampling of the environment of chemistry and biological hazard.In addition, magnetic-particle can with catch the part and candidate drug compounds locate altogether.Can catch the interaction of cell and fixed medical compounds to the further analysis of catching of interested cell.Therefore, device can be used for from composite mix isolated cell subgroup and measure them and the reactivity of candidate drug compounds to be used for high-throughout pharmacy drug discovery process and based on the screening of the candidate compound of secondary cell.In other embodiments, the receptor-ligand binding research that is used for drug discovery can realize in flowing on the magnetic-particle and in the composite mix of candidate ligand (or agonist or antagonist) by will to catch part be receptor mapping at device.Interested part is hunted down and binding events can be detected, for example by using the secondary dyeing of fluorescent probe.This embodiment makes it possible to identify not existing or existing of known ligand or identify candidate drug compounds from the composite mix of being extracted by tissue or cell dissociation thing fast.

Magnetic-particle.The selective retention of analyte can obtain by magnetic-particle being introduced (for example be connected to be present in the obstacle in the device or to be processed into to produce and increase the long-pending obstacle of analyte interactive surfaces to increase the bonded possibility) in the device of the present invention.Catching part can be in conjunction with magnetic-particle to influence the specificity combination of target analyte.In another embodiment, magnetic-particle can be processed into and make the analyte that only allows to have selected size, shape or deformability pass through device.Also considered the associating of these embodiments.For example, device can be configured to keep based on some analyte of size with based on other analytes of bonded.In addition, device can be related to becoming in conjunction with surpassing a kind of interested analyte, for example order, the parallel or zone arranged or wherein two or more are caught and partly are configured on the same magnetic particle or on the adjacent particles, for example in conjunction with identical obstacle or zone of scattering in device.In addition, can specificly a plurality ofly catch certain applications on identical or different magnetic-particle with having in the device, for example be disposed on the identical or different obstacle or zone for same analyte (for example anti-CD71 and anti-CD36).

Flow condition in the device is normally like this: promptly very gently the analyte in the treatment unit destroys preventing.From the positive pressure of fluid column or negative pressure pump or flow and can be used for analyte is transported into or transports microfluidic device of the present invention.This device can make and soft processing maximize the frequency that each analyte and one or more magnetic-particle collide simultaneously.Target analyte when colliding with magnetic-particle and any part of catching interact.Because the design result of magnetic attraction is caught partly and can be located altogether with obstacle in the device.This interaction causes catching and keeping target analyte in qualifying part.Can discharge the analyte of catching by the magnetic regions degaussing that will keep magnetic-particle.Discharge from partial selectivity for analyte, degaussing can be confined to selected obstacle or zone.For example, magnetic field can be designed to have electro permanent magnetic, makes it possible to start arbitrarily and close the magnetic field of each individual region or obstacle.In other embodiments, can and catch key release particles partly by the failure analysis thing, for example by chemical chop or destruction non-covalent interaction, or by reducing the magnetic response of bonded analyte.For example, some ferrous particle is connected with monoclonal antibody through the DNA connexon, and the use of DAN enzyme can and discharge analyte from ferrous particle cutting.Alternatively, antibody crack protein enzyme (for example papain) can be used for processing selecting release.Increase also can be used for particularly discharging magnetic-particle in the hard magnetic zone from magnetic regions for the shearing force of magnetic-particle.In other embodiments, do not discharge the analyte of catching, and can when the analyte of catching keeps, analyze or further operation it.

Figure 76 has described and has been designed to catch and the embodiment that separates the bank of expressing the cell that changes the iron acceptor from composite mix.The monoclonal antibody of the CD71 acceptor of covalent coupling magneticsubstance obtains stock easily, such as but not limited to doping ferrous polystyrene and iron particle or iron colloid (for example from Miltenyi and Dynal).Monoclonal antibody in conjunction with the CD71 of magnetic-particle flows into bank.The particle of coated antibody is attracted to obstacle (for example post), bottom and wall by the magnetic field interaction force between particle and the magnetic field and keeps.By the particle between flushing (adjustable commutating speed make away from the hydrodynamic shear on the particle of obstacle greater than magneticstrength) obstacle with by lax those that keep of spheroid away from the local magnetic field influence of obstacle.

Figure 77 be to use full Transferrins,iron complexes with bank be used for from composite mix for example blood catch and discharge the preferred embodiment of CD71+ cell.The iron level of full Transferrins,iron complexes is abundant, and commercially available acquisition has higher affinity costant and specificity interactional with it than its corresponding monoclonal antibody to the CD71 acceptor.Be used as following dual purpose with the iron of Transferrins,iron complexes ligand coupling: keep and the conformation of cell receptor bonded part and the molecule paramagnetic element of the part on the conduct reservation obstacle.

Enrichment

In one embodiment, device of the present invention has been used to produce enrichment the sample of required analyte, for example to small part based on magnetic and optional hydromeehanics size.The application of such enrichment comprises concentrating analysis, for example comprises the particle of rare cells.Device also can be used for for example organoid (for example nucleus) of enrichment of cell component.Ideally, apparatus and method of the present invention keep at least 1%, 10%, 30%, 50%, 75%, 80%, 90%, 95%, 98% or 99% required analyte with respect to original mixture, potential enrichment simultaneously is with respect to one or more non-required analytes at least 1,10,100,1,000,10,000,100,000 or even 1,000,000 times required analyte.Although the required analyte in the sample increases with respect to the concentration of other analytes, enrichment also can cause required analyte to dilute with respect to primary sample.Preferably, extent of dilution is at the most 90%, for example at the most 75%, 50%, 33%, 25%, 10% or 1%.

In another embodiment, device of the present invention has been used to produce enrichment the sample of rare analyte.Common rare analyte is with 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% or 0.000001% analyte that exists that is less than all analytes in the sample or its quality 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% or 0.000001% analyte less than the sample total mass.Per sample, exemplary rare analyte comprises fetal cell, medullary cell, progenitor cell, stem cell (for example undifferentiated), foam cell, cancer cells, immune system cell (host or graft), epidermic cell, endotheliocyte, endometrial cell, trophoblast, phoirocyte, bacterium, fungi, virus and pathogenic agent (for example cell or protozoon).Rare analyte like this can comprise for example originate pathogen isolation the water sample for example of blood or environment of body fluid from sample.Fetal erythrocyte can for example be used for definite for example developmental sex of fetus and evaluation dysploidy or hereditary property and for example suddenly change from the maternal peripheral blood enrichment.Circulating tumor cell, normally epidermic cell type or source also can be from the peripheral blood enrichment to be used to diagnose the purpose with the monitor therapy progress.The circulation endothelium cell can also be similarly from the peripheral blood enrichment.

Also can be to body fluid or environmental sample screening pathogenic agent, for example septicemia or bacterium or viral meningitis of intestinal bacteria, blood borne disease for example.Rare cells also comprises the cell from an organism that is present in another organism, for example from the cell of transplant organ.

The blood that extracts or the amount of other body fluid can be according to Mammals and for example conceived section of situation thereof or disease for example cancer and differences.In certain embodiments, obtain to be less than 50mL, 40mL, 30mL, 20mL, 10mL, 9mL, 8mL, 7mL, 6mL, 5mL, 4mL, 3mL, 2mL, 1mL, 0.5mL, 0.1mL, 0.05mL or even the fluid of 0.01mL from individuality.In certain embodiments, obtain the fluid of 1-50mL, 2-40mL, 3-30mL or 4-20mL from individuality.In certain embodiments, to surpass 5,10,15,20,15,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or the fluid of 100mL from individuality.For example, in certain embodiments, system and method for the present invention allow from the maternal blood sample detection that is less than 5mL or 3mL with separate rare cells (for example fetal cell).In other embodiments, system and method for the present invention can be used for analyzing or enrichment from relatively large blood for example greater than 20mL or surpass those the rare cells of 50mL.Any one of above-mentioned function can for example be less than generation in 1 day or 12,10,11,9,8,7,6,5,4,3,2 or 1 hours.Collected intact sample can be applicable to the device that the present invention is used for enrichment and/or detects rare cells.Alternatively, can handle sample like this, make that only some component is introduced into device.

Except the enriching rare analyte, device can be used for preparing purposes.The illustrative preparation purposes comprises from blood generation cell pack.In one embodiment, device can be configured to separately or the enrichment of associating determinacy produces part with thrombocyte, red corpuscle and white corpuscle enrichment by magnetic resolution.By using multichannel or multi-stage type device, all three kinds of cell parts can produce from same sample parallel or in proper order.In other embodiments, device can be used for for example separating karyocyte from Cord blood from cytode.

Therein by the analyte of enrichment through be damaged or the situation of other degradeds in, device of the present invention is favourable.As described herein, device can be designed to minimum collision enrichment of analyte (for example cell) between analyte and obstacle or other surfaces.This minimizes the physical disturbance that has reduced analyte (for example cell), and in the situation of cell, also prevents or reduce interior activation of cell that collision causes.In the situation of cell, soft handle limited amount rare analyte in the protection sample and prevent from or reduce to cause component in the cell to be polluted or the breaking of degraded, and prevent or reduce cell for example stem cell or hematoblastic maturation or activation.In preferred embodiments, enrichment of analyte so promptly is less than 30%, 10%, 5%, 1%, 0.1% or even 0.01% destroyed (for example activating or mechanical lysis).

Figure 33 has shown that the typical sizes of cell in the human peripheral blood distributes.Leukocytic scope is～4 μ m are to～18 μ m, and red corpuscle is～1.5 μ m (minor axis).Be designed to separate white corpuscle and erythrocytic determinacy device has holding back size and passing through size greater than the maximum of 18 μ m of 2 to 4 μ m usually.Such device can be united magnetic resolution as herein described and be used.

Figure 57 A has shown the operation of the determinacy device that is used for the enrichment purpose.Sample inlet by device adds cell sample, and adds buffer solution medium by fluid intake.Cell less than critical size does not move to deflection by device, occurs from the edge outlet in their primary sample medium.Greater than the cell of critical size for example epidermic cell be deflected, and occur from central outlets, be included in the buffer solution medium that adds by fluid intake.Therefore, the operation of device produces to be greater than or less than the sample of critical size enrichment.Because among the maximum cell of epidermic cell in blood flow, the size in gap of device and geometrical shape can be selected in fact every other cell type is imported the edge outlet, simultaneously from central outlets be created in single by after the device basically with the sample of epidermic cell enrichment.

The determinacy device that the present invention includes does not need to form diad shown in Figure 57 A, with operation as described herein.Signal figure representation diad device or single array shown in Figure 57 B.

Can be in many ways by magnetic resolution and determinacy separation coupling be strengthened enrichment.For example, pearl (for example immune affine pearl) labels targets analyte (for example cell) be can use, thereby their size (describing) and the magnetic of also potential change analyte increased as Figure 59.In the situation of epidermic cell, the size that this can also increase them causes even more effective separation.Alternatively, the size of less analyte (for example cell) can be increased to such degree, promptly they become in the sample maximum target or with occupy unique size range with respect to other components in the sample, or make they and other analyte copurifications.Pearl can maybe can be adhered to any other material preparation of analyte (for example cell) by polystyrene, magneticsubstance.Ideally, such pearl has medium buoyancy and makes and can not destroy flowing of labeled cell by device.The change of analyte size can be used for parallel or use in order based on magnetic and deterministic enrichment.

Change

In other embodiments, except enrichment or under the situation of no enrichment, can make interested analyte with can chemistry or physics change in the sample analyte or fluidic and change reagent and contact.Its application comprises purifying, buffer exchange, mark (for example immunohistochemistry, magnetic and histological chemistry's mark, cell dyeing, and mobile fluorescence in situ hybridization (FISH)), magnetic change, cell fixation, cytotostatic, lysis and cell-stimulating.

Such method can allow from sample analyte to be transferred to different liqs (for example buffer exchange).Figure 34 A shows this graphic unistage type determinacy device that influences, Figure 34 B has shown this effect of multi-stage type determinacy device, Figure 34 C has shown this effect of the diad array of determinacy device, and Figure 34 D has shown this effect of the multi-stage type diad array of determinacy device.Similarly, magnetic resolution is used in retention analysis thing in the device, or with required direction deflection it, to produce buffer exchange.By using such method, but the analyte in the enriched sample (for example hemocyte).The transfer of analyte from a kind of liquid to another kind of liquid also can be used for producing a series of changes, for example the auspicious special Albert'stain Albert of blood on the wafer.A series of changes like this can comprise make analyte and first reagent react and then analyte is transferred to lavation buffer solution and then with another kind of reagent react.

Figure 35 A-35C has described another embodiment that changes in the two-section type determinacy device with two by-pass channels.In this embodiment, insection 1, will move to damping fluid (reagent that for example contains the analyte magnetic that changes) and collection from blood than big analyte, insection 2 analyte of intermediate sizes is moved to damping fluid (reagent that for example contains the analyte magnetic that changes) from blood, and also be collected in the less analyte that Duan Zhongwei is moved from blood.Figure 35 B has described two sections size and has held back, and Figure 35 C has described the distribution of sizes of three parts of collecting.Then, collected part can stand the enrichment based on magnetic.

Figure 36 has described the embodiment that changes in the two-section type determinacy device with the edge, side that is configured in array and the by-pass channel between the conduit wall, for example the embodiment that changes of magnetic.Figure 37 has described the determinacy device similar to Figure 36, except connecting two sections by the fluid channel.Figure 38 has described the change of using little trace in the determinacy device with two sections.Figure 39 A-39B has described wherein output terminal change in the captive device in single passage of first section and second section.Figure 40 has described the another kind of device that is used for the inventive method.

Figure 41 has described the determinacy device and has been used to implement change to a plurality of orders of analyte.In this device, from sample analyte is moved to reagent with analyte response, and then the analyte that changes is moved to damping fluid, thereby remove unreacted reagent or byproduct of reaction.Can increase other step (step for example as herein described).

Enrichment and change also can be united.For example, required cell is contacted with lytic reagent, and based on size, magnetic or the two enrichment of cell component nucleus for example.In another embodiment, the particulate mark that makes analyte and bound analyte for example magnetic bead contact.Can remove unconjugated particulate mark based on size, magnetic or the two.

Concentrate

Device of the present invention can also be used for for example interested cell of concentrating sample.In an embodiment shown in Figure 62, cell sample is introduced the sample inlet of determinacy device.The volume of the damping fluid by reducing to introduce fluid intake makes this volume significantly less than the volume of cell sample, produces the concentration of the target cell of smaller size smaller.Similarly, in passage, keep the magnetic response analyte and can be used for concentrating analysis, for example by discharge the analyte that keeps with smaller size smaller.This enrichment step can improve the result of any downstream analysis of being implemented.

Lysis

Device of the present invention also can be used for the purpose of lysis.In order to realize this purpose in the determinacy device, follow and the similar scheme that is used for enrichment: the sample inlet (Figure 63) by device adds cell sample, and adds lysis buffer by fluid intake.As mentioned above, be deflected and enter lysis buffer, cause these lysis greater than the cell of critical size.Therefore, the sample that occurs from central outlets comprises the cracked cellular component, comprises organoid, and undeflected full cell occurs from another outlet.Similarly, can make the cell that is retained in the device based on magnetic contact, for example with component in the cell that discharges magnetic bonded analyte with lytic reagent.Therefore, described device provides the method for a kind of selective splitting target cell.

Downstream analysis

Can use any method known in the art to detect the analyte of enrichment, for example rare cells and/or component.For example, in certain embodiments, detection components of the present invention comprises imager, for example microscope, Kamera, spectrometer or Hyper spectral Imaging device (seeing for example Vo-Dinh etc., IEEE Eng.Med.Biol.Mag.23:40-49 (2004)).Detection can relate to the detection of preferential painted use and colour-change, and it is pointed out the existence of interested analyte or does not exist.In certain embodiments, be used for the indirect immuning dyeing method of dyeing application of policies that cell is identified, it is the secondary antibodies that the enzyme yoke closes then that this method is used unlabelled primary antibody.Exemplary enzyme comprises horseradish peroxidase and alkaline phosphatase.The dyestuff that produces look results from the colourless substrate of known conjugase.In certain embodiments, the rare cells to painted evaluation uses specific probe further to analyze chromosome abnormalty by nucleic acid hybridization.This screening strategy preferably uses a kind of dyestuff to be used for the rare cells not isolabeling relevant with nucleic acid probe.

The key prerequisite that many diagnostic are measured is to remove from the free or unreacted change reagent of sample to be analyzed or be decreased to be lower than it below threshold level.In one embodiment, described reagent is labelled reagent.As mentioned above, device of the present invention can be analyzed the labelled reagent (for example cell) of free labelled reagent and bound analyte.Then, implement the volumetric measurement of reaction reagent and the background interference do not had from the conspicuous level of labelled reagent is possible.In one embodiment, use to the specific epidermal cells mark for example EpCAM have optionally fluorescence antibody.Fluorescence part can comprise that Cy dyestuff, Alexa dyestuff or other contain the molecule of fluorophor.The enrichment of analyte that uses photofluorometer to produce by detecting then to have mark is the mark sample that fluorometric analysis produced of the sample of cell for example.Alternatively, the mark that contains fluorophor can be united the spectrometer use.The detected value that is obtained can be used for to the quantity of the target analyte in the sample for example cell carry out quantitatively.

Many other detection methods and labelled reagent can be used for method and apparatus of the present invention.Traget antibody can occupy the covalently bound enzyme that cuts substrate, changes its absorbancy in setted wavelength, uses spectrometer that the degree of cutting is carried out quantitatively then.According to the substrate that uses, colourity or luminous reading are possible.Advantageously, the use of enzyme labelling allows the signal of remarkable augmentation detection, reduces the threshold value of detectability.

Quantum dot is for example from QuantumDot company

Can be used as covalent attachment and catch for example labelled reagent of antibody of part.Qdot has resistivity to photobleaching, and can unite two photon excitations and detect use.

The possible labelled reagent that another kind can be used for the inventive method is a phage.Phage display is a kind of wherein by target for example being present in the technology that binding peptide is showed in engineering phage strain that protein on the interested cell has strong avidity.Coding is corresponding to the peptide sequence of given phage in the nucleic acid of given phage.Therefore, phage is useful labelled reagent, therefore this is to think that they may for example cell be little and can be separated easily than analyte, and they also carry and can use PCR or similar technique analysis and quantitative nucleic acid, make it possible to the quantity that is present in the cell in the enriched sample is carried out quantitative assay.

Figure 65 has described phage as labelled reagent, and wherein two determinacy device sections are arranged with cascade configuration.The method that Figure 65 describes meets the general description of Figure 64, except using labelled reagent.Applied magnetic enrichment similarly.

Downstream analysis can comprise the accurate mensuration of required analyte (for example cell) quantity in the analyzed sample.In order to produce quantitative result accurately, the quantity of the target of labelled reagent (surface antigen on for example interested cell) usually necessary known or measurable (for example expression level in the gene cell), and the combination of labelled reagent also should be carried out in measurable mode, for example noiseless material.Therefore, it is particularly useful to produce the apparatus and method of highly enriched cell sample before introducing labelled reagent.In addition, in the situation of the required analyte (for example epidermic cell in the blood sample) of rareness, the preferred labelled reagent that allows the amplification of signal that produces.Allow the reagent of amplification of signal to comprise enzyme and phage.Other labelled reagents that do not allow to increase easily but produce strong signal for example quantum dot also are desirable.Changing or unlabelled analyte also takes place quantitatively.

When apparatus and method of the present invention are used for cell that enriched sample contains, can to mutually on the same group cell implement further quantitatively and molecular biological analysis.The cell that the described volumetric measurement method of coupling is softly handled in apparatus of the present invention has been kept cell integrity, makes can implement further analysis if desired.For example, can implement to destroy the technology of cell integrity after volumetric measurement, such technology comprises DNA or RNA analysis, Proteomic analysis or intermediate metabolites analysis.An example of such analysis is PCR, wherein the level of the lysing cell and the specific dna sequence dna that increases.When the quantity of isolating target cell when very low, such technology is particularly useful.

Cancerous diagnose

In the patient's who suffers from mammary cancer, intestinal cancer, liver cancer, ovarian cancer, carcinoma of the pancreas and lung cancer circulation, found the epidermic cell that strips off from solid tumor.Usually, the existence of treatment back circulating tumor cell (CTC) is with tumour progression and diffusion, reduce to be associated to Low Response, palindromia and/or the survival rate for the treatment of.Therefore, the calculating of CTC provides a kind of method that patient's baseline characteristic is classified, this method indication initial risk and based on to the reaction of treatment continue after risk.

But it is different with the tumour derived cell in dormancy and the long-life marrow, the CTC of epidermis the types and sources has about 1 day short-half-life, and their existence prompting is from the up-to-date stream (Patel etc., Ann Surg, 235:226-231,2002) of outgrowth tumour.Therefore, CTC can reflect the present clinical state and the therapeutic response of patient disease.The counting of CTC and being characterized in is estimated in cancer prognosis and monitor therapy effect can cause palindromia with early detection the treatment failure has potential value.In addition, CTC analyzes the early stage recurrence that can detect among the presymptomatic patient who has finished the course of treatment, the individuality of no detectable disease is unsuitable for participating in the clinical trial (Braun etc., NEngl J Med, 351:824-826,2004) of new treatment likely at present.

Apparatus and method of the present invention can be used for estimating the cancer patients and being in those of suffering from the cancer risk.For example, extract blood sample and be introduced into apparatus of the present invention from the patient with separating table chrotoplast and other hemocytes.Detect the quantity of blood sample mesocuticle cell, for example use method as herein described.For example, can use antibody labeling cell, and antibody can have covalently bound fluorescent mark in conjunction with EpCAM, or in conjunction with magnetic-particle.Then, can carry out volumetric measurement to the enriched sample that described device produces, and detect, can measure the quantity of the epidermic cell that exists in the initial sample of blood from this.Microscopy can be used for estimating quantitative cell so that the respective numbers of the labeled cell in volumetric measurement and the blood sample is associated.

By carrying out a series of detections, can follow the tracks of the level that is present in the epidermic cell in patient's blood flow as the function of time through a couple of days, several weeks, several months or several years.In the patient's who has cancer situation, this useful prompting of progression of disease is provided and help the doctor according to the increase of for list chrotoplast in patient's blood flow, reduce or do not have to change and carry out suitable treatment and select.For being in those of risk of cancer, the unexpected increase of the cell quantity that is detected can provide the patient to develop the early warning of tumour.This early diagnosis and continue after the treatment intervention may cause improved patient result with respect to no diagnostic message.

Diagnostic method comprises the volumetric measurement of carrying out from the label table chrotoplast of blood separation, and the technology of destroying the cell integrity.For example, Auele Specific Primer that can be by using the particular cancer mark is implemented PCR to the low-down sample of quantity of isolating target cell wherein, can obtain the information about the tumor type of the origin of cell analyzed.In addition, the method for one or more types of the cancer that RNA analysis, Proteomic analysis or intermediate metabolites analysis can be existed among the patient as diagnosis and implementing.

A kind of important diagnostic index of lung cancer or other cancers is some sudden change that has or do not exist EGF-R ELISA (EGFR).EGFR comprises the extracellular ligand binding domains, strides membrane portions and intracellular tyrosine kinases (TK) structural domain.The normal physiological effect of EGFR is to comprise Urogastron (EGF) at the extracellular combining site in conjunction with the ErbB part, to excite signal cascade in the cell of downstream, causes cell proliferation, survival, motion and other correlated activations.Many nonsmall-cell lung cancer response small molecules EGFR inhibitor, for example Gefitinib (gefitinib) (Iressa (Iressa) with EGFR sudden change; But normal final obtain to make their drug-fast secondary mutations AstraZeneca).Use apparatus and method of the present invention, can be by obtaining frequent blood sample and detecting as the quantity of each sample mesocuticle cell of the function of time and monitor the patient who uses such medicine.For example, for list chrotoplast alleviates and the size of one or more tumours reduces through the quantity of time decreased prompting disease seriousness.And then epidermic cell quantitatively after, by these cells of pcr analysis with the EFGR that determines to express in the epidermic cell based in have which kind of sudden change.Known some sudden change for example around the EGFRTK structural domain accumulative those make cancer cells suppress responsive to Gefitinib.Therefore, the existence support of these sudden changes may respond the diagnosis of the cancer of the treatment of using Gefitinib.Yet telomutation finally takes place in many patients of response Gefitinib, often is that 790 methionine(Met) to Threonines at the extron 20 of TK structural domain replace, and makes them have resistance to Gefitinib.The apparatus and method of the application of the invention can also detect this sudden change, this provide about the further diagnostic message of the course of disease with and the possibility of response Gefitinib or similar compound.

Fetal cell detects

Apparatus and method as herein described can be applicable to from the conceived human blood sample that obtains, with the illness or the abnormal conditions of for example screening fetus.When the screening fetus, can be from conceived Mammals in conceived 24,20,16,12,10,8 or 4 weeks or the conceived human blood sample that obtains.In other embodiments, after stopping, pregnancy can carry out the screening and the detection of fetal cell.

For example, in certain embodiments, by the antigen of dyeing detection rare cells, for example ε/γ globin (cytoplasmic), GPA, i antigen, CD71 or its combination.The combination of ε and γ globin is found in the fetal nucleated red blood (fNRBC ' s) of the 95-100% in conceived 10-24 week.Al-Mufti etc., (2001) Haematologica 85,357-362; Choolani etc., (2003) Mol.Hum.Reprod., 9,227-235.This ε-γ combination or separately the γ globin be used for fNRBC is dyeed Bohmer for example, (1998) Br J Haematol.103,351-60.; Choolani etc., (2003); Christensen etc., (2005) Fetal Diagri.Ther.20,106-112; And Hennerbichler etc., (2002) Cytometry, 48, described in the 87-92.As seen be less than 10 false positive among each fNRBC, have or do not have the CD71 enrichment, make that therefore globin is (＞10, the 000 times) letter sorting (triage) with high degree of specificity.The antibody of two kinds of globins is that those skilled in the art are known.Dyeing can cause as the scoring of the binary of positive or negative or cause providing the varying strength of antigen amount in the analyte.

Glycophorin A and CD71 are the other antigen that can be used for detecting cell type.GPA is present in all red corpuscle pedigrees.Therefore, it can be used for detecting erythroblast, and no matter their sudden change level how.Think that GPA is found on the red corpuscle pedigree cell only, be found in usually on the considerably less circulating cells, and there is increase in it in conceived process.The FACS sorting is presented at the combination of CD71 and GPA in the conceived process and is present at least 0.15% the monocyte, Price etc. for example, (1991) Am.J.Obstet Gynecol, 165,1713-1717; Sohda etc., (1997) Prenat.Diagn., 17,743-752.

Antigen i also can be used as the mark that separates and/or detect fetal cell, Sitar etc. for example, (2005) Exp.Cell Res., 302,153-161.Nineteen fifty uses patient's polyclonal serum to describe antigen i first.Continue after the antigen " I " of two kinds of forms of digital proof or " i " on adult and fetal cell, express respectively.

In case detect fetal cell or interested component, can further analyze them for multiple purpose, for example sex or hereditary situation.In certain embodiments, the analysis of fetal cell or its component is used for determining the existence of genetic abnormality or do not exist that for example karyomit(e), DNA or RNA are unusual.The example of autosomal chromosome abnormalty includes but not limited to Angleman syndrome (15q11.2-q13), cat's cry syndrome (5p-), DiGeorge syndrome and Velo-cardiofacial syndrome (22q11.2) Miller-Dieker syndrome (17p13.3), PW (15q11.2-q13), retinoblastoma (13q14), Smith-Magenis syndrome (1,7p1 1.2), 13 trisomes,trisomy 16,18 trisomes, trisomy 21 (mongolism), triploidy, williams syndrome (7q1 1.23) and Wolf-Hirschhorn (4p-).The example of sex chromosomal abnormality include but not limited to kallman syndrome (Xp22.3), steroid sulfuric ester defective (STS) (Xp22.3), the chain ichthyosis of X-(ichthiosis) (Xp22.3), Klinefelter syndrome (XXY), Fragile X syndrome, super-female or X trisome and X monomer.

Other can include but not limited to disappearance (little disappearance part), micro-deleted (trace disappearance material can only comprise single-gene), transposition (a chromosomal part is connected with another karyomit(e)) and inversion (a chromosomal part is wiped out and reversing insertion again) by the more uncommon chromosome abnormalty of systems analysis of the present invention.

In certain embodiments, the analysis of fetal cell or its component is used to analyze SNP and based on the situation of such SNP prediction fetus.

In any embodiment of this paper, can use any method known in the art to detect/analyze.The example that is used to detect/analyze the method for abnormal conditions include but not limited to karyotyping, in situ hybridization (ISH) (for example fluorescence in situ hybridization (FISH), produce chromogen position hybridization (CISH), nanometer gold in situ hybridization (NISH), restriction fragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR) technology, flow cytometry, Electron Microscopy, quantum dot, and the nucleic acid array that is used to detect single nucleotide polymorphism (SNP) or rna level.In certain embodiments, be used to detect two or more methods of genetic abnormality.For example, multiple FISH probe or other dna probes can be used for analysis list cell or interested component.

The enrichment of cell subsets

Method and apparatus of the present invention also can be used for the enrichment of cell subgroup.For example, in ripening process, erythroblast changes form and hemoglobin concentration.Based on the difference of hemoglobin concentration, the karyocyte in separable late period is eosinophilic normoblast and early stage erythroblast, for example rubricyte for example.Figure 99 has shown the nRBC in the different stage of maturity of using the methods described herein acquisition.

Erythrophagocytosis

Method and apparatus of the present invention also can be used for the cell that enrichment has the red corpuscle of including in (erythrophagocytosis).Figure 100 has shown to have the RBC that includes in and be retained in monocyte in the magnetic devices of the present invention.Erythrophagocytosis is relevant with unusual illness with multiple disease, for example familial hemophagocytic histiocytosis, acute monocytic leukemia and lymphoma.The validity that the detection of these cells can be used for diagnosing particular disorder or determines treatment.For example, method of the present invention can be used for the quantity of cell with the RBC that includes in is counted.

Described method also can be generally used for including in or in conjunction with magnetic component magnetotactic bacteria any cell of magnetotactic water spirillum (Aquaspirillum magnetotacticum) for example for example.Magnetotactic bacteria also can be by other cytophagies, and these cells can use method enrichment of the present invention then.

The enrichment of erythroblast

The level of circulation nRBC raises and is associated with multiple disease and unusual illness, for example erythroleukemia, β-major thalaseemia, BartShi oxyphorase and immune hemolytic anemia.Allow enrichment nRBC or their nucleus based on magnetic and size or based on the combination of cracked enrichment.The validity that the detection of these cells can be used for diagnosing particular disorder or determines treatment.

The combination of enriching method

Any enriching method as herein described can order or the enrichment that is used to produce interested analyte simultaneously, for example cell or nucleus.When application combination, can implement them with desired sequence usually.For example, described method can comprise separation, magnetic resolution and full cell or the selective splitting based on size.As described herein, described method also can comprise based in conjunction with catch the part for example antibody affine enrichment or based on the spectral quality of cell granulations or the enrichment of other detectability matter.

The device that is used to implement such enrichment combination can comprise the separation assembly that is used for each enrichment, single device that respectively makes up or install or be used for various enrichments, two or more types enrichment can take place, and for example shifts by suitable fluid or the artificial or robot that depends on sample between the device and connects.

Specimen preparation

Sample can be handled or be used for method as herein described with being untreated, and is for example stable and remove some component.In one embodiment, before sample is introduced apparatus of the present invention with interested analyte cell enrichment sample for example.The method that is used for the enrichment of cell group has at this paper to be described and is known in the art, for example based on the enrichment of affine mechanism, magnetic, aggegation and size, shape and deformability.Can dilute or concentrate it before with some sample introduction device.

Preferably, sample for example blood from 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours, 2 hours that obtain or be applied to system within an hour.In certain embodiments, when when the patient extracts blood sample, be applied to system of the present invention.Preferably, under 4-37 ℃ temperature with sample application in system.

In one embodiment, reagent is added into the hydromeehanics size of sample with analyte in selectivity or the non-selective increase sample.Then, for example pump the sample of this change by the determinacy device.Because the particle swelling also has the hydromeehanics size of increase, it is possible that use has big determinacy device with the gap size that is easier to make, in a preferred embodiment, implement swelling and based on the step of size enrichment in complete mode in the determinacy device.Suitable reagent comprises any hypotonic solution, deionized water for example, 2% sucrose solution, or pure non-aqueous solvent.Other reagent comprise selective binding (for example by antibody or avidin-vitamin H) or non-selective bonded pearl, for example magnetic beads or polymeric beads.

In another embodiment, reagent is added into sample with selectivity or non-selectively reduce particulate hydromeehanics size in the sample.The inhomogeneous difference that reduces to increase hydromeehanics size between the particle of particulate in the sample.For example, ooze contractive cell by height and analyze karyocyte and cytode.Cytode is retractable into very little particle, and karyocyte can not be contracted to and is lower than nuclear size.Exemplary contraction reagent comprises hypertonic solution.

In an optional embodiment, affine functionalized pearl is used for increasing the size of the interested analyte of other analytes that exist with respect to sample, thereby allows operation to have determinacy the device big and gap size that is easier to make.

As described herein, the change of such size can with based on the enrichment of magnetic order or parallel application.

When obtaining sample for example during blood sample, can in the container that contains one or more following reagent, collect: stablizer, sanitas, fixing agent, cracking agent, thinner, anti-apoptotic agent, antithrombotics, antithrombotics, buffer reagent, osmotic pressure regulator, pH regulator agent, change the reagent and/or the linking agent of magnetic.

Can be by device active or passive actuating fluid.Can use electric field, centrifuge field, pressure-actuated fluid stream, electroosmotic flow or capillary action to pump fluid.In preferred embodiments, Chang mean direction is parallel with conduit wall.

Following any exemplary determinacy apparatus and method can be incorporated device of the present invention into.

Embodiment

Embodiment 1: the silicon device of multichannel 14 syllogic array diads

Figure 42 A-42E has shown the exemplary means with following feature.

Dimension: 90mm * 34mm * 1mm

Array design: three sections, the gap size of first, second and the 3rd section is=18,12 and 8 μ m respectively.Bifurcation ratio=1/10.Diad, single by-pass channel

Device design: multichannel 14 array diads, the resistance to flow device of stability of flow

Device assembling: use standard lithography and degree of depth pasc reaction etching technique that array and passage are assemblied in the silicon.Etch depth is 150 μ m.The hole that passes through that fluid enters uses the KOH wet etching to make.The pressure sensibility goo of use blood compatibility (9795,3M, St Paul MN) is encapsulated in silicon matrix the fluid channel of sealing with formation on the etching face.

Device package: will install with having and send blood and damping fluid to device and the plastic composite pipe that extracts the external fluid bank of the part that produces and engage.

The device operation: external pressure source is used for the pressure application of 2.4PSI is sent and extracted to regulate from the fluid of the device of packing in damping fluid and blood bank.

Experiment condition: will be collected into K from the human blood of adult's donor of volunteer₂EDTA vacuum vessel (vacutainers) (366643, Becton Dickinson, Franklin Lakes, NJ).In 9 hours of extracting, use above-mentioned exemplary means to handle undiluted blood (Figure 42 F) in room temperature.Separate to come the karyocyte and the cytode (red corpuscle and thrombocyte) of autoblood, and blood plasma is delivered to contains 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, DulbeccoShi phosphate buffered saline (PBS) (the 14190-144 of no calcium MO) and magnesium, Invitrogen, Carlsbad is in damping fluid stream CA).

Detection technique: use Coulter impedance blood analyser (Ac-T diff^TM, Beckman Coulter, Fullerton CA) detects complete blood count.

Performance: Figure 43 A-43F has shown the typical histogram that is produced from the blood sample that produced of device and refuse (damping fluid, blood plasma, red corpuscle and thrombocyte) and product (damping fluid and karyocyte) part by blood analyser.Table 1 has shown the performance of 5 kinds of different blood samples.

Table 1

Sample number	Treatment capacity		Performance	Tolerance
							RBC removes	Platelet removal	TheWBC loss
1	4mL/hr	100％	99％	＜1％
					2	6mL/hr	100％	99％	＜1％
3	6mL/hr	100％	99％	＜1％
					4	6mL/hr	100％	97％	＜1％
5	6mL/hr	100％	98％	＜1％

The silicon device ofembodiment 2multichannels 14 unistage type array diads

Figure 44 A-44D has shown the exemplary means with following feature.

Dimension: 90mm * 34mm * 1mm

The array design: 1 section, gap size=24 μ m.Bifurcation ratio=1/60.Diad, two by-pass channels

Device design: multichannel 14 array diads, the resistance to flow device of stability of flow

Device assembling: use standard lithography and degree of depth pasc reaction etching technique that array and passage are assemblied in the silicon.Etch depth is 150 μ m.The hole that passes through that fluid enters uses the KOH wet etching to make.The pressure sensibility goo of use blood compatibility (9795,3M, St Paul MN) is encapsulated in silicon matrix the fluid channel of sealing with formation on the etching face.

Device package: will install with having and send blood and damping fluid to device and the plastic composite pipe that extracts the external fluid bank of the part that produces and engage.

The device operation: external pressure source is used for the pressure application of 2.4 PSI is sent and extracted to regulate from the fluid of the device of packing in damping fluid and blood bank.

Experiment condition: will be collected into K from the human blood of adult's donor of volunteer₂The EDTA vacuum vessel (366643, Becton Dickinson, Franklin Lakes, NJ).In 9 hours of extracting, use above-mentioned exemplary means to handle undiluted blood in room temperature.Separate to come the karyocyte and the cytode (red corpuscle and thrombocyte) of autoblood, and blood plasma is delivered to contains 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, DulbeccoShi phosphate buffered saline (PBS) (the 14190-144 of no calcium MO) and magnesium, Invitrogen, Carlsbad is in damping fluid stream CA).

Detection technique: use Coulter impedance blood analyser (

Ac-T diff^RM, Beckman Coulter, Fullerton CA) detects complete blood count.

Performance: be installed on operation in 17mL/ hour and obtained＞99% red corpuscle removes,＞95% karyocyte keeps, and＞98% platelet removal.

Embodiment 3: the separation of fetal cord blood

Figure 45 has shown the schematic representation of apparatus that is used to separate karyocyte and fetal cord blood.

Dimension: 100mm * 28mm * 1mm

Array design: three sections, the gap size of first, second and the 3rd section is=18,12 and 8 μ m respectively.Bifurcation ratio=1/10.Diad, two by-pass channels.

Device design: multichannel 10 array diads, the resistance to flow device of stability of flow.

Device assembling: use standard lithography and degree of depth pasc reaction etching technique that array and passage are assemblied in the silicon.Etch depth is 140 μ m.The hole that passes through that fluid enters uses the KOH wet etching to make.The pressure sensibility goo of use blood compatibility (9795,3M, St Paul MN) is encapsulated in silicon matrix the fluid channel of sealing with formation on the etching face.

Device package: will install with having and send blood and damping fluid to device and the plastic composite pipe that extracts the external fluid bank of the part that produces and engage.

The device operation: external pressure source is used for the pressure application of 2.0 PSI is sent and extracted to regulate from the fluid of the device of packing in damping fluid and blood bank.

Experiment condition: human foetus's Cord blood is extracted to the phosphate buffered saline (PBS) that contains the ACD antithrombotics.In 48 hours of extracting, use said apparatus in room temperature to handle 1mL blood in 3mL/ hour.Separate to come the karyocyte and the cytode (red corpuscle and thrombocyte) of autoblood, and blood plasma is delivered to contains 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, DulbeccoShi phosphate buffered saline (PBS) (the 14190-144 of no calcium MO) and magnesium, Invitrogen, Carlsbad is in damping fluid stream CA).

Detection technique: the cell smear of preparation product and waste part (Figure 46 A-46B) also uses the Wright-Giemsa of modification (WGl 6, Sigma Aldrich, St.Louis, MO) dyeing.

Performance: in product part (Figure 46 A), observe fetal nucleated red blood, and do not observe at waste part (Figure 46 B).

Embodiment 4: from the maternal blood isolation of fetal cells

Unite the feasibility that is used to prove from the maternal blood isolation of fetal cells with executing apparatus and method and the affine beneficiation technologies of immune magnetic described in detail in the example 1.

Experiment condition: after the pregnant volunteer's that male fetus arranged parent donor is selected termination of pregnancy, immediately with its blood collecting to K₂The EDTA vacuum vessel (366643, Becton Dickinson, FranklinLakes, NJ) in.In 9 hours of extracting,use embodiment 1 described device to handle undiluted blood in room temperature.Separate to come the karyocyte and the cytode (red corpuscle and thrombocyte) of autoblood, and blood plasma is delivered to contains 1% bovine serum albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, DulbeccoShi phosphate buffered saline (PBS) (the 14190-144 of no calcium MO) and magnesium, Invitrogen, Carlsbad is in damping fluid stream CA).Afterwards, (Auburn CA) is marked with karyocyte part and use MimMACS for 130-046-201, Miltenyi Biotech Inc. to use anti-CD71 nano-beads according to manufacturer's specification sheets^TMMS post (130-042-201, Miltenyi Biotech Inc., Auburn, CA) enrichment.At last, with CD71 male portion branch to slide glass.

Detection technique: (Abbott Laboratories, Downer ' s Grove IL) use fluorescence in situ hybridization (FISH) technology that the slide glass of a mistake is dyeed to use the Vysis probe according to manufacturer's specification sheets.Sample is colored because of having X and Y chromosome.In a kind of situation, the sample from the pregnant tire preparation of known trisomy 21 is also carried out No. 21 chromosomal dyeing.

Performance: confirmed the separation of fetal cell by the reliable existence of the male sex cell from the positive group of CD71 of karyocyte part (Figure 47) preparation.In the one abnormal conditions that detected, also identified trisomy 21 pathology (Figure 48).

Embodiment 5-10 has shown the specific embodiment of device.The quantity of the section of order, the gap size of each section, the quantity (array/wafer) of ε (flow angle) and each device passage are provided for the description of each device.Use DRJE to assemble out each device, and each device have thermal oxide layer from silicon.

Embodiment 5

This device comprises 5 sections of single array.

The array design: 5 sections, asymmetric array

Gap size: section 1:8 μ m

Section 2:10 μ m

Section 3:12 μ m

Section 4:14 μ m

Section 5:16 μ m

Flow angle: 1/10

Array/wafer: 1

Embodiment 6

This device comprises several sections, and wherein each section is the diad with by-pass channel.Height of devices is 125 μ m.

Array design: symmetry syllogic array with central collection channel

Gap size: section 1:8 μ m

Section 2:12 μ m

Section 3:15 μ msection 4

Section 5

Flow angle: 1/10

Array/wafer: 1

Other: central collection channel

Figure 49 A has shown the covert that is used for assembling device.Figure B1B-B1D is the augmenting portion that limits the covert of inlet, array and outlet.Figure 50 A-50G has shown the SEM of actual device.

Embodiment 7

This device comprises several sections, and wherein each section is the diad with by-pass channel." fin " is designed to the adjacent by-pass channel of side to keep entering array again from the fluid of by-pass channel.Wafer also comprises the resistance to flow device on the wafer, and promptly entrance and exit has the fluid resistance greater than array.Height of devices is 117 μ m.

Array design: syllogic symmetric array

Gap size: section 1:8 μ m

Section 2:12 μ m

Section 3:18 μ m

Section 4: section 5:

Flow angle: 1/10

Array/wafer: 10

Other: resistance to flow device on the central collection channel wafer of big fin

Figure 51 A has shown the covert that is used for assembling device.Figure 51 B-51D is the augmenting portion that limits the covert of inlet, array and outlet.Figure 52 A-52F has shown the SEM of actual device.

Embodiment 8

This device comprises several sections, and wherein each section is the diad with by-pass channel." fin " is designed to the adjacent by-pass channel of side to keep entering array again from the fluid of by-pass channel.The shape of analog array will be become near the edge designs of the fin of array.Wafer also comprises the resistance to flow device on the wafer, and promptly entrance and exit has the fluid resistance greater than array.Height of devices is 138 μ m.

Array design: syllogic symmetric array

Gap size: section 1:8 μ m

Section 2:12 μ m

Section 3:18 μ m

Section 4:

Section 5:

Flow angle: 1/10

Array/wafer: 10

Other: resistance to flow device on the big fin of the alternate central authorities collection channel wafer

Figure 45 A has shown the covert that is used for assembling device.Figure 45 B-45D is the augmenting portion that limits the covert of inlet, array and outlet.Figure 53 2A-532F has shown the SEM of actual device.

Embodiment 9

This device comprises several sections, and wherein each section is the diad with by-pass channel.Using Femlab to optimize " fin " makes the adjacent by-pass channel of side to keep entering array again from the fluid of by-pass channel.The shape of analog array will be become near the edge designs of the fin of array.Wafer also comprises the resistance to flow device on the wafer, and promptly entrance and exit has the fluid resistance greater than array.Height of devices is 139 or 142 μ m.

Array design: syllogic symmetric array

Gap size: section 1:8 μ m

Section 2:12 μ m

Section 3:18 μ m

Section 4:

Section 5: flow angle: 1/10

Array/wafer: 10

Other: resistance to flow device on central collection channel (Femlab I) wafer that Femlab optimizes

Figure 54 A has shown the covert that is used for assembling device.Figure 54 B-E1D is the augmenting portion that limits the covert of inlet, array and outlet.Figure 55 A-55S has shown the SEM of actual device.

Embodiment 10

This device comprises a kind of single hop diad device, and it has the by-pass channel that is configured to receive from the end of two arrays output terminal.Obstacle in this device is oval.Molded array border in Femlab.Wafer also comprises the resistance to flow device on the wafer, and promptly entrance and exit has the fluid resistance greater than array.Height of devices is 152 μ m.

Array design: unistage type symmetric array

Gap size: section 1:24 μ m

Section 2:

Section 3:

Section 4:

Section 5: flow angle: 1/60

Array/wafer: 14

Other: the molded array boundary of resistance gauge Femlab on the central obstacle cylindroid wafer

Figure 44 A has shown the covert that is used for assembling device.Figure 44 B-44D is the augmenting portion that limits the covert of inlet, array and outlet.Figure 56 A-56C has shown the SEM of actual device.

Embodiment 11

The determinacy device of incorporating apparatus of the present invention into can pass through computer aided design (CAD) (CAD) design and pass through the little assembling of photolithography.Open two step method, wherein at first blood sample has been removed bulk (debulked) removing a large amount of minicells, and recovered rare target epidermic cell target cell by immune affinity capture then.By the photolithography device for limiting and based on the design that CAD produces it is etched in the silicon matrix.The cell enrichment assembly of approximate test slide glass size contains parallel sample processing part and is connected to common sample and cushions inlet and the sample preparation passage that is associated of product and Waste outlet.Each several part contains the obstacle array of little assembling, and described array is optimized to through moving than maxicell to product stream and by the apart target cell type.In this embodiment, microwafer is designed to separating red corpuscle (RBC) and thrombocyte and bigger white corpuscle and circulating tumor cell.Target cell group from the whole blood collection enrichment by device.Estimate the performance (Figure 67) of cell enrichment microwafer by separating RBC and thrombocyte and white corpuscle (WBC) in the normal whole blood.In the cancer patients, partly find circulating tumor cell in bigger WBC.Blood is carried out bottom line dilution (30%), and handle the 6ml sample with the flow velocity that reaches 6ml/ hour.In automatic distinguishing, dimensioning and count different hemocyte groups' Coulter " A^c-T diff " estimate product and waste streams in the clinical haemanalysis instrument of type.The enrichment wafer has been realized separating of RBC and WBC, wherein WBC partly have＞99% karyocyte keeps,＞99% RBC remove and＞97% thrombocyte removes.The typical histogram of these cell parts is shown among Figure 68.Conventional cytolgical examination confirms highly enriched (Figure 69) of WBC RBC part.

Next, in using the functionalized microfluid component of fixed antibody, collect epidermic cell by affinity capture.Designed the capture component of single chamber of the regular array that has the little assembling obstacle that comprises antibody sandwich.These obstacles are configured to make cell capture maximization, its by increase approximate four times catch area and by the stream of cells the laminar flow of the adjacent barrier that slows down under with the duration of contact between increase cell and the fixed antibody.Can avoid damage with the protection cell at operation capture component at high relatively flow velocity but under the condition of low-shearing power.By using 10% silane, 0.5% glutaraldehyde (gluteraldehyde) and avidin is that biotinylated anti-EpCAM subsequent treatment comes to carry out functionalized to the surface of capture component afterwards.Be used in the 3% bovine serum albumin sealing reactive site among the PBS, use the Tris HCl of dilution to quench, and use the L Histidine of dilution stable.After each section, use among the PBS cleaning assembly and at last in drying at room temperature and storage.Use human advanced lung cancer clone NCI-HI 650 (ATCC CRL-5883) to detect acquisition performance.This clone has the in-frame deletion of heterozygosis 15bp in the exons 19 of EGFR, make it to the Gefitinib sensitivity.Use the trypsinase results to converge the cell of culture, use vital dye Cell Tracker Orange (CMRA reagent, Molecular Probes, Eugene, OR) dyeing is resuspended in the fresh whole blood and with different in flow rate and carries out classification in the microfluid wafer.In these initial feasible experiments, directly in capture component, handle cell suspending liquid, and need not in the cell enrichment assembly, carry out classification in advance red corpuscle is removed bulk, therefore, sample flow contains normoerythrocyte and white corpuscle and tumour cell.Be to handle in the capture component after the cell, use the buffer solution for cleaning device to remove the cell of non-specific binding in high flow velocities (3ml/ hour).Remove adherent top, and use Paraformaldehyde 96 that adherent cell is fixed on the wafer, and pass through fluorescence microscope.Reclaim from blood-counter system counting average cell, typically catch and the results are shown in table 2.Initial productive rate in the reconstruct of unassorted blood research is greater than 60%, has to be less than 50% non-specific binding.

Table 2

Loop No.	Mean flow rate	Cycling time	The cell count of handling	The capturedcell number	Yield
							1	3.0	1 hour	150,000	38,012	25％
2	1.5	2 hours	150,000	30,000/ml	60％
						3	1.08	2 hours	108,000	66,661	64％
4	1.21	2 hours	121,000	75,491	62％

Next, the original position successful analysis by application of sample to the whole blood and NCI-Hl 650 cells that reclaim by size classification and affinity capture as mentioned above.In distinguishing epidermic cell and leukocytic test round, the mother liquor of the full white corpuscle monoclonal antibody of the fluorescently-labeled CD45 of 0.5ml is sent in the capture component, and inroom temperature incubation 30 minutes.Use the buffer solution for cleaning assembly removing unconjugated antibody, and use 1% Paraformaldehyde 96 with cell fixation on wafer and pass through fluorescence microscope.Shown in Figure 70, epidermic cell is bonded to the bottom of obstacle and capture component.Obviously because low-level non-specific catching can be observed the background dyeing of CD45 pan-leukocyte antibody to flow passage, as several white corpuscles that are colored.

Embodiment 12

The device embodiment

Preferred determinacy Design of device is shown in Figure 73 A, is shown in Figure 73 B corresponding to three preferred parameters of installing embodiments relevant with this design.These embodiments are particularly useful for from the blood separation epidermic cell.

The PCR of embodiment 13 EGFR sudden change measures

Processing is from cancer patients's blood sample and use the apparatus and method of embodiment 11 to analyze, and has produced the enriched sample of the epidermic cell that contains CTC.Analyze this sample then to identify potential EGFR sudden change.

In order to implement this analysis, target cell isolation of genomic DNA from the sample that is present in enrichment and amplification are used for the allele-specific PCR in real time and measure.Because the known coding region that all EGFR sudden changes of Gefitinib susceptibility or resistance NSC lung cancer is positioned at exons 18 to 21 of giving of report uses unique group of exon Auele Specific Primer that these four exons are carried out pcr amplification separately so far.Next, use TaqMan 5 ' nuclease mensuration PCR system (Applied Biosystems) and 7300 type Applied Biosystems PCR in real time machines to carry out the reaction of multichannel allele-specific quantitative PCR.This allows any known clinical relevant sudden change of Rapid identification.

This method needs two step PCR schemes.Exons 18 to 21 at first increases in Standard PC R reaction.The PCR product that is produced is divided into each equal portions to be measured to be used for allele-specific multichannel PCR in real time.In the logarithmic phase process, stop initial PCR reaction so that in the amplification procedure allele-specific information loss may be minimum.Next, second subprovince of taking turns the specific initial p CR product of each interested sudden change of pcr amplification.Supposing that PCR in real time has very high susceptibility, also is possible even separate few complete information that obtains the sudden change situation of EGFR gene to 10CTC.PCR in real time provide with 8 logarithmic input DNA concentration carries out the allelotrope sequence quantitatively, therefore in addition the heterozygous mutant among the assorted group also can use this method easily to detect.

Use primer Optimization Software program Primer Express (Applied Biosystems) design oligonucleotides, and optimize hybridization conditions to distinguish Wild type EGFR dna sequence dna and mutant allele.From the known lung cancer cell line that carries EGFR sudden change H358 (wild-type), H1650 (15-bp disappearance for example, Δ 2235-2249) and H1975 (two point mutation, 2369C → T, 2573T → G) the EGFR genomic dna of amplification is used to optimize the allele-specific real-time PCR reactions.Use TaqMan5 ' nuclease, developed the specific alleles specific marker probe of wild-type sequence or known EGFR sudden change.Be about to oligonucleotide and be designed to have the temperature of fusion of easy differentiation coupling and mispairing, and the PCR in real time condition optimizing is become to distinguish wild-type and mutation allele.All real-time PCR reactions have been carried out with three parts.

At the beginning, the label probe that will contain wild-type sequence in the reaction identical with the single mutation probe carries out the multichannel processing.The result is expressed as a mutant allele sequence can identifies the sample that contains or do not have given sudden change with respect to the ratio of wild-type sequence.After the condition of having optimized given probe groups, all mutant alleles that multichannel is handled in the given exon in identical PCR in real time is measured are possible then, and this has increased the simplification of using this analysis tool in clinical setting.

The purity of the input sample of CTC can be different, the sudden change situation of isolating CTC can be xenogeneic.However, the high susceptibility of PCR in real time makes it possible to identify any and mutant nucleotide sequences all existence.

Embodiment 14 measures the counting of non-epidermic cell type

Use method of the present invention, can be based on the counting of the cell type except epidermic cell is diagnosed.The diagnosis that does not exist, exists or make progress of cancer can be based on greater than the cell quantity in the specific cell sample of holding back size.For example, can select to have the cell of 14 microns fluid cell dias.This holds back size can remove most of white corpuscles.Measure the character of these cells then by downstream molecules or cytological analysis.

The cell type except epidermic cell of available analyses comprises endotheliocyte, endothelial progenitor cells, endometrial cell or the trophoblast that can point out morbid state.In addition, the counting separately of measuring epidermic cell and other cell types is measured the diagnostic message that ratio between the quantity of the quantity of epidermic cell and other cell types can provide usefulness then.

The determinacy device can be configured to separate target cell subgroup, for example above-mentioned those.For example shown in Figure 71 A-D.Selectable size is held back and is made most of natural hemocytes comprise that red corpuscle, white corpuscle and thrombocyte flow to refuse, is collected in the sample of enrichment but not n cell comprises endotheliocyte, endothelial progenitor cells, endometrial cell or trophoderm parent cell.Can further analyze the sample of this enrichment.

Therefore, using the determinacy device, is possible based on size from blood or other body fluid isolated cell subgroups, and this allows to remove most natural hemocyte easily when target maxicell type.As the graphic demonstration of Figure 72, the determinacy device can comprise the count tool of measuring the cell quantity in the enriched sample, and the further analysis of cell can provide and can be used for diagnosing or other information of other purposes in the enriched sample.

Embodiment 15 is from maternal blood enrichment fetal nucleated red blood

For this embodiment, as described herein, described device comprises can the isolating fetal erythroblast and the determinacy separation assembly of parent white corpuscle and parent erythroplastid.This determinacy assembly is connected with the bank that contains Sodium Nitrite.With in the maternal blood sample introduction device that has diluted to produce with the fetal erythrocyte enrichment and to remove the erythrocytic part of parent.Thereby this sample is imported its Central Asia hour sodium oxidation fetal hemoglobin iron to be increased in the bank of magnetic response of fetal erythrocyte.Then, apply magnetic field through for example MACS post, the fetal erythrocyte of change is in conjunction with magnet, and the parent white corpuscle is not by the magnet combination.Remove white corpuscle and remove magnetic field then by resin for example and allow to reclaim fetal erythrocyte, for example analyze, store or further operate being used for.

Embodiment 16 uses high gradient magnet from the blood separation fetal nucleated red blood

Can be used for attracting to contain among graphic Figure 78 of being shown in of erythrocytic exemplary high gradient magnet of methemoglobin.Because the inhomogeneous character in the magnetic field that is applied places the red corpuscle of kapillary to be condensed to separate areas (Figure 79 A-79C).

Figure 80 is from erythroblast (positive part) precipitation of male Cord blood preparation and the picture of white corpuscle precipitation (negative part).At first use determinacy side tripping device to extract karyocyte, and use the Sodium Nitrite of 50M to handle 10 minutes from blood.Make karyocyte pass through the magnetic post then, erythroblast keeps in the magnetic post.In post, magneticstrength is about 1 tesla, and field gradient is about 3000 teslas/m, and flow velocity is about 0.4mm/sec.The Dulbecco PBS damping fluid that use contains 1%BSA and 2mM EDTA washes out white corpuscle and it is collected as negative part from post.Use same buffer with flow velocity collect as positive part from post wash-out erythroblast and with it.

Figure 81 is to use method that Figure 80 the describes a series of fluorescence photos from the isolating erythroblast of maternal blood.Use fluorescence in situ hybridization (FISH) pair cell to dye.Use the water gauge note probe of α SAT-zone to identify X chromosome respectively, use the redness in α satellite and satellite III district and green stain to identify X chromosome.Use DAPI (blueness) pair cell nuclear to redye.

Figure 82 has shown that the method for using Figure 80 description is from the maternal blood erythroblast in isolating different stage of maturity.Use Wright-Giemsa dyeing pair cell to dye.

Figure 83 A and 83B have shown method (B) the enrichment result's who uses anti-CD71 antibody (A) and Figure 80 description Photomicrograph.Sample among the A contains＞200,000 karyocytes from 1mL blood, and the every mL blood of the sample among the B contains 100-500 the karyocyte of having an appointment.The purity of the erythroblast that the method for describing by Figure 80 obtains surpasses about 1000 times based on the enriching method of antibody.

Figure 101 has shown the Photomicrograph of the fetal cell of the trisomy 21 that the order enrichment used in the determinacy device and magnetite gathering obtain.

The illustrative methods of embodiment 17 enrichment of cell

In affine enrichment, the sample that makes as described herein is by the determinacy device.The output of determinacy device is contacted with the magnetic bead of coated antibody or other selective binding part.Magnetic resolution is in conjunction with the cell of pearl then, and cell rotates, and analyzes, and for example passes through FISH.In the oxyphorase enrichment, the sample that makes as described herein is by the determinacy device.The output that makes the determinacy device then and reagent that can oxygenated haemoglobin for example Sodium Nitrite contact, and magnetic resolution magnetic response sexual cell.Can carry out the cell rotation to isolated cells, and analyze, for example, maybe can carry out analysis of molecules by FISH.In complete method, the inventive system comprises determinacy enrichment assembly and magnetite gathering assembly, its output can stand analysis of molecules.Figure 85 has shown the synoptic diagram of intact device of the present invention.

Selective splitting after embodiment 18 enrichments

Although the following example concentrates on the cell nuclear colony that extracts the circulation fetal cell from the parent whole blood, described method is common for the cellular component that separates other cells.

The separation of fetal cell nuclear

Figure 86 has shown from the schema of the method for maternal blood sample separation fetal cell nuclear.This method causes erythrocytic preferential cracking (Figure 87).

The several embodiments that are used for genome analysis from the nucleus purifying group of the interested circulating cells of separation of whole blood are described below:

A) as described herein, this method comprises the microfluidic process of whole blood, with 1) by removing the enriched sample that 1 to 3 logarithmic erythroblast and hematoblastic quantity produce karyocyte, 2) residual erythroplastid, seedless reticulocyte and the preferred nuclear parent of the erythroblast white corpuscle of cracking discharges fetal cell nuclear by microfluidic process enrichment the nuclear sample being arranged, 3) from parent the nuclear leukocyte separating nucleus is arranged by device microfluidic process, and 4 based on size) use the genetic analysis tool analysis fetus genome of commercially available acquisition.

B)step 1 and 2 that this method is designed toembodiment 1 is integratedly by microfluidic device, uses downstream unit then or carries out step 3 (seeing Figure 88 and 89) than the assembly of bigger device.Figure 88 shown be used to produce occur together based on the enrichment of size and the synoptic diagram of cracked microfluidic device.This device has been used two zones of obstacle, described obstacle deflection from the device edge than maxicell, wherein sample is introduced in the centre gangway that contains cracked solution (diad device for example as herein described).For maternal blood, barrier region is processed into and makes erythroblast and thrombocyte be retained in the edge of device, and fetal erythrocyte and other karyocytes are deflected to centre gangway.In a single day fetal erythrocyte (interested cell) is deflected in the centre gangway, promptly cleaved.Figure 89 has shown the synoptic diagram based on the microfluidic device of size that is used for from uncracked cellular segregation nucleus (interested cellular component).This device is similar to Figure 88's, make nucleus be retained in the edge of device except obstacle is processed into, and bigger particle is deflected to centre gangway.

C) integrated processes of microfluid based on the generation of fetal cell nuclear in the maternal blood sample be then the batch processing technology for example density gradient centrifugation with isolation of fetal cells nuclear and mother cell (seeing Figure 90).

D) selective splitting of method and principle evidence erythroblast with separate.Use the hypotonic and ammonium chloride cracking of two kinds of methods to come the red corpuscle that pollutes in the donor blood sample of the pregnant Cord blood application of sample of cracking foot.Because the erythroblast cracking is faster than cytode in hypotonic solution, the exposure duration of mixed cellularity group will cause the difference cracking based on the cell mass of this time in the control hypotonic solution.In the method, deposition of cells forms precipitation, and draws the blood plasma on the precipitation.Add deionized water then, and will precipitate with water and mix.The exposure in 15 seconds is enough to the erythroplastid of cracking＞95%, has MIN erythroblast cracking, the exposure in 15 to 30 seconds be enough to cracking＞70% erythroblast but＜other karyocytes of 15%, and＞will increase other karyocyte cracked percentage ratio 30 seconds.After required exposure duration, add 10 * HBSS (height oozes balanced salt) solution the condition of oozing such as solution is returned to.Be exposed to the ammonium chloride solution red corpuscle that cracking is large quantities of of normal concentration (for example 0.15M isotonic solution) and karyocyte is had MIN influence.When the osmotic pressure with cracked solution was reduced to the hypotonic ammonium chloride solution of generation, large quantities of erythroblasts quilts were with the mature erythrocyte cracking.

The density centrifugation method is used to obtain the lymphocyte populations of enrichment.These lymphocytes of portion are exposed to the cell of hypotonic ammonium chloride solution time enough with cracking＞95%.Use these nucleus of specific stain Hoechst 33342 (Bb H 33342) mark in the rich district of AT of double-stranded DNA then, and its time is added into the primary lymphocyte populations to produce 90: 10 (cells: mixture nucleus).Mixture is supplied to as Figure 89 described, collects refuse and product part, and measure the cell that contains in the each several part: the nucleus ratio based in apart cell and the nuclear device.

The density gradient centrifugation of split product.Can be by sucrose pad solution be added in the lysate, the cracked nucleus of the cell mixing suspension of difference cracking routine processes has been passed through in enrichment.This mixture is laid on the pure sucrose pad solution then, and the nucleus of centrifugal then formation enrichment precipitation.Draw uncracked cell and fragment from supernatant liquor, the nucleus pellet resuspended in buffered soln, and is rotated to its cell on the slide glass then.

Total lysis of acid alcohol and nRNA FISH that target cell is identified.Will be from be exposed to acid alcohol solution (acetate methanol 3: 1v/v) 30 minutes, cause＞99% cytode cracking and＞99.0% karyocyte cracking on ice as the described product that obtains based on the device of apart cell of Figure 89.Also can comprise by with cellular exposure in salts solution (0.6% NaCl) 30 minutes with the nuclear hypotonic processing of swelling before the acid alcohol cracking.Can the nucleus that discharge quantitatively be positioned on the slide glass by cell rotation and FISH (Figure 95 a and 95b).Can use the positive select ζ sphaeroprotein for example, ε sphaeroprotein, gamma Globulin and negative select pin for example the spy of the beta Globulin length using RNA-FISH or analyze telomere identify interested cell, for example fetal nucleated red blood.Other methods that are used to distinguish fetus and non-fetal cell are known in the art, and for example U.S. Patent No. 5,766, and 843.

Embodiment 19 single hops are based on the device of size

Figure 91 has shown and has been optimized in the separating blood particulate based on the device of size.It is the fixed interval (FI) width with 22 μ m, has the single hop device of 48 multichannel arrays that are used for the parallel sample processing.The parameter of this device is as follows:

The array design:	L5
		Gap size:	Section 1:22 μ m
Flowing angle:	1/50
		Array/wafer	48
Rated depth	150μm
		The device footprint	30mm×64mm
DESIGNED FEATURE	The by-pass channel stability of flow of the single array optimization of multichannel flows to supply with and flow and extracts the border

Obtain blood from volunteer's donor of pregnancy, and use (iDPBS) dilution in 1: 1 of DulbeccoShi phosphate buffered saline (PBS) (no calcium and magnesium).Use the active pressure of 0.8PSI that blood and electrophoretic buffer (iDPBS that contains 1%BSA and 2mM EDTA) are delivered in the device asembodiment 20 described joint multiple-unit tubes.Blood is separated into two-pack: the karyocyte in electrophoretic buffer and cytode in damping fluid and plasma proteins.Use the normal impedance calculating instrument to analyze this two kinds of components.Using iodine third pyridine dyeing solution combination with standard Nageotte counting chamber to characterize the component that contains karyocyte in addition loses with definite total karyocyte.Collected data are used for determining the reservation of blood processing volume (mL), blood treatment rate (mL/ hour), the hematoblastic removal of RBC/ and karyocyte.Following form provides the result who uses this device cell enrichment:

Handle volume (mL)	26.5	8	15.4	17	19
						Treatment capacity (Ml/h)	10.6	10.0	11.8	9.8	9.8
WBC/ input WBC (Nageotte) in the refuse	0.013％	0.012％	0.005％	0.014％	0.030％
						RBC removes	99.993％	99.992％	99.997％	99.995％	99.999％
Platelet removal	99.6％	99.7％	99.7％	99.7％	99.7％

Embodiment 20 is used for the multiple-unit tube based on the device of size

The exemplary multiple-unit tube that microfluidic device of the present invention inserts is shown in Figure 92.Between having, places multiple-unit tube the two halves of microfluidic device of the present invention.Half of multiple-unit tube comprises the inlet that separates of blood and damping fluid, and each inlet is connected with corresponding fluid bank.Passage in the device is oriented to and makes their holes in device connect bank.Usually, device is vertically oriented, and is collected when handled droplet of blood goes out the product outlet.Zone around the product outlet of microfluidic device also can use hydrophobic substance for example to carry out the size of mark with the drop of restriction formation from the hydrophobic substance of permanent marking.Device also comprises two hydrophobic venting port strainers, for example the PTFE strainer of 0.2 μ m.The air that these strainers allow to catch in devices is shifted by the aqueous solution, but do not allow liquid with low pressure for example＜5psi passes through.

For initiating device, under reduced pressure and simultaneously stir the DulbeccoShi PBS that damping fluid is for example contained 1% bovine serum albumin (w/v) and 2mM EDTA and outgased 5-10 minute.The damping fluid inlet of pressure in multiple-unit tube with＜5psi pumps into device with damping fluid then.Then, damping fluid is filled the damping fluid chamber by hydrophobic venting port strainer displaced air, and fills passage and blood chamber in the microfluidic device then.The hydrophobic venting port strainer that is connected with blood chamber allows the displacement of air in the chamber.In case blood chamber is filled, damping fluid promptly is pumped to the blood inlet, and in certain embodiments, after causing 1 minute with 1psi, folder closes the blood inlet, and pressure boost continues 3 minutes to 3psi.

Hypotonic cracking after embodiment 21 enrichments

By add a large amount of low ionic strength buffer liquid for example deionized water and the selective splitting nucleus that nuclear RBC and nRBC arranged and discharge them make the fetus nRBC group of device enrichment described herein stand hypotonic shock.Then, stop hypotonic shock by adding isopyknic high ionic strength buffers liquid.The nucleus of the release of for example gathering in the crops by the Visipaque 320 aqueous solution (p=1.32g/mL) by gradient centrifugation after analyzing.

Figure 93 has described the selective splitting with respect to parent nRBC as the fetus nRBC of the function of the time length that is exposed to cracking condition.This selective splitting process also can be used in the cell mass that selective splitting fetus nRBC forms fetus nRBC, nuclear fetus and parent RBCs and fetus and parent white corpuscle are arranged.Use distilled water induce hypotonic shock to continue the given time period and add then isopyknic 10 * salts solution for example PBS to stop it, fetus nRBC and parent nRBC cracking in time, cracked in this time course (unvital) fetus nRBC increases to 10 times, and cracked parent nRBC increases less multiple.At any given time point, use iodine third pyridine that the cracked cell is dyeed, and concentrate to determine the ratio of cracked fetus nRBC with respect to parent nRBC by gradient centrifugation.The time length of optimizing can be determined and be used for selective enrichment fetus nRBC nucleus.

The selective splitting ofembodiment 22 mother cells

Fetus nRBC group by any device enrichment as herein described can stand the erythrocytic selective splitting of parent.

For the seedless RBC of selective splitting and parent have nuclear RBC, handle the sample of fetus nRBC enrichment as follows: use for example 0.155M NH of RBC lysis buffer₄Cl, 0.01MKHCO₃2mM EDTA, contain carbonic anhydrase inhibitor for example the 1%BSA of acetazolamide (for example at 0.1-100mM) to induce cracking, use then and contain ion-exchange channel inhibitor 4,4 '-two isothiocyano benzene-2,2 '-a large amount of balanced salt damping fluids of disulfonic acid (DIDS) for example the 1xPBS of 10x volume or balanced salt damping fluid for example 1xPBS stop cracking.Then, make the fetal cell of survival stand the selection and the analysis of other rounds.

Use Tonostan and fluorexon AM mark to stimulate leukocytic K562 cell (Figure 94) in room temperature.The K562 cell of these marks is added into blood sample, adds damping fluid (0.155M NH then₄Cl, 0.01M KHCO₃, 2mM EDTA, 1%BSA, and 10mM acetazolamide) and (ratio of the blood volume of damping fluid volume and application of sample is 3: 2).In the soft blood sample that stirs incubation application of sample down ofroom temperature cycle 4 hours.Measure vigor cell part in the sample of each application of sample by measuring green fluorescence in 610nm at a plurality of time points.Only observation of cell cracking (under the situation at no DIDS) after handling 3 minutes.

Embodiment 23 genetic analyses

Cleavable by for example any device as herein described or method with the sample of fetus nRBC enrichment and analyze its hereditary inclusion.The lysis of required cell or cellular component and isolating possibility method comprise:

A) can make sample stand total lysis to remove tenuigenin and separating nucleus with fetus nRBC enrichment.Can by use stationary liquid for example the CarnoyShi stationary liquid handle and adhere to slide glass and fix nucleus.Can through the immunostaining of pair cell nucleoprotein and transcription factor or through differential hybridization fetus premessenger RNA RNA FISH's and identify fetal cell nuclear (Gribnau etc., Mol Cell 2000.377-86 by the existence of endogenous fetus target; Osborne etc., Nat Gene.2004.1065-71; Wang etc., Proc.Natl.Acad.Sci.1991.7391-7395; Alfonso-Pizarro etc., NucleicAcids Research.1984.8363-8380.).These endogenous fetus targets can comprise sphaeroprotein, ζ sphaeroprotein, ε sphaeroprotein, gamma Globulin, δ sphaeroprotein, beta Globulin, alpha globulin and non-sphaeroprotein target I-q enzyme (Yu etc. for example for example, Blood.2003101:2081), N-acetyl-glucosamine transferring enzyme or IgnT.The oligonucleotide probe that RNA FISH uses can be intron-exon border or other regional oligonucleotide probes, the length that it can be identified required target uniquely or pass through to analyze telomere;

B) can useembodiment 22 described damping fluids and ion-exchange inhibitor selective splitting with the sample of fetus nRBC enrichment with isolation of fetal cells.Can be again by cell inner mark for example sphaeroprotein and the β of I-branch 1,6-N-acetylglucosamine based transferase or surface markers for example antigen I existence or do not exist the fetal cell that makes survival to stand to select.In another embodiment, as described inembodiment 22, can make the fetus nRBC of enrichment stand selective splitting, use antibody to carry out the lysis of complement-mediated then at CD45 (be present in all surface antigen in the nuclear leukocyte is arranged) to remove seedless RBC and parent nRBC.The complete fetus nRBC that is produced should not have any other contamination of cells;

C) as described in the embodiment 21, can examine with isolation of fetal cells with the sample of fetus nRBC enrichment by the hypotonic shock cracking.By use stationary liquid for example the CarnoyShi stationary liquid handle and adhere to slide glass and fix nucleus.

In case separate required cell or cellular component (for example nucleus), can analyze its hereditary inclusion.FISH can be used for identifying karyomit(e) 13 and 18 or defective or other chromosome abnormalties for example trisomy 21 or XXY.For example also can using, the method for comparative genome hybridization detects chromosomal aneuploidy.In addition, the fetal cell that can use micro-dissection to check.When extracting, can make the nucleic acid of fetal cell stand one or take turns PCR or complete genome amplification carries out comparative genome hybridization then, or short series connection repeats, and (STR) analyze, for example mononucleotide site mutation (SNP), disappearance or transposition of gene mutation analysis more.

Embodiment 24 is based on the cracking after the enrichment of size

Use 50mM Sodium Nitrite/0.1mM acetazolamide to handle and comprise the erythrocytic product of 3ml in1xPBS 10 minutes as acquisition as described in Figure 89.Make cell and 0.155M NH then₄Cl, 0.01M KHCO₃2mM EDTA, the contact of the lysis buffer of 1%BSA and 0.1mM acetazolamide, and by directly splashing into the ion-exchanger channel inhibitor for example 4 that containsBAND 3,4 '-two isothiocyano benzene-2,2 '-quenching solution of disulfonic acid (DIDS) stops scission reaction.To seedless RBC with there is the RBC of examining to count, and FISH is used for fetus nRBC is counted after Wright-Giemsa dyeing.Then numerical value and uncracked contrast are compared.Such experimental result is shown in as follows:

	Before the cracking	After the cracking	Cell rate of recovery %
				eRBC	2.6×106	0.03×106	～1％

nRBC	42	26	62％
				fnRBC
	6	4	68％

The enrichment ofembodiment 25 apoptotic DNA

Total cracking of chaotropic salt (Chaotropic salt) or washing agent mediation and the oligonucleotide mediated enrichment that the apoptosis DNA of nuclear RBC is arranged from fetus.For example buffered guanidine hydrochloride solution (4.0M at least), guanidine thiocyanate (4.0M at least) or buffered washing agent solution for example contain the product of cracking from obtaining as the described device of Figure 89 in the tris buffer of SDS in chaotropic salt solution.Then in 55 ℃ with cell lysate with the Proteinase K incubation of 10 μ l 50mg/ml to remove protein, then in 95 ℃ of incubations 5 minutes with inactivated proteases.When fetus nRBC enters the maternal blood circulation time, carry out apoptosis, and this apoptosis process causes the dna break of fetus nRBCDNA.By utilization reduce size fetus nRBC DNA with use oligonucleotide mediated enrichment more efficient to separate the less dna fragmentation of complete genome group DNA, can by with solution in is connected with pearl or comes the apoptotic fetus nRBC of selective enrichment DNA for example to lack the repetition (STR) of connecting with the molecule marker of identifying uniqueness with the oligonucleotide hybridization of array or other surface bonding.After hybridization, can use for example undesired nucleic acid of 150mM sodium-chlor flush away or other pollutents in 10mM Tris HCL (pH 7.5) of high salt buffer solution, then the target of catching is released into damping fluid for example in 10mM Tris pH 7.8 or the distilled water.Then, thus use as the embodiment 23 described apoptotic DNA that are used for the methods analyst enrichment of analyzing gene group inclusion.

Embodiment 26 cracking programs

Figure 96 has shown the schema of describing the cracking change of program that can implement the maternal blood sample in detail.Although for example use the sample of the enrichment of apparatus and method generation as herein described to begin to describe from the sample of enrichment, can implement this method to any maternal blood sample.This chart has been described and can have been carried out the cell (for example fetal cell) that cracking needs with (i) selective splitting; The (ii) cell that needs of selective splitting and their nucleus; (iii) cracking all cells; (iv) cracking all cells and their nucleus; (v) cracking unwanted cells (for example parent RBC, WBC, thrombocyte or its combination); (vi) cracking unwanted cells and their nucleus; And (the vii) nucleus of cracking all cells and selective splitting unwanted cells.This chart has also shown nuclear illustrative methods (apparatus and method of the present invention can be used for this purpose) that is used to separate release and the method that is used for measurement result.

The titration of embodiment 27 full lysises

This is the embodiment of the full lysis of titration in microfluidic environment.As herein described based on apart and the blood sample of enrichment is divided into 4 parts of equal-volumes with using.Make three parts of volumes by microfluidic device, described microfluidic device can transport cells be used to limit the first predefine medium of path length to the device, is transported to the second predefine medium that is used to collect then.Make volumetric cell suspension flow velocity difference, to allow being controlled at the contact second predefine medium before along the time of the path length that limits with the first predefine medium incubation, in this embodiment, DI water is as the first predefine medium, and 2 * PBS is as the second predefine medium.Adjust flow velocity, make in DI water incubation time of 10,20 or 30 seconds cell to be mixed, with 2 * PBS with the generation isotonic solution.Use blood-counter system to calculate total cell count of 3 volumes that are untreated of handling volume and being left.

Sample	The beginning cell counting	Last cell counting	% keeps
				1 handles	6.6×106	6.6×106	100％
2 exposed for 10 seconds	6.6×106	7.2×105	10.9％
				3 exposed for 20 seconds	6.6×106	4.6×105	6.9％
4 exposed for 30 seconds	6.6×106	3.4×105	5.2％

The device of embodiment 28 magnetite gatherings

This embodiment provides a kind of and has used single magnetic post to put with the low specificity example luggage that separates magnetic cell of dividing a word with a hyphen at the end of a line.Use the marked by magnetic bead target cell or use for example NaNO of chemical reagent₂Be translated into paramagnetism.

The figure of this device is shown in Figure 97, and its photo is shown in Figure 98.This device comprises the magnetic post, connects the fluid bank of pillar one end, the valve that the fluid of the control pillar the other end connects, flow control apparatus, buffer container, and waste fluid container.

This device can followingly use:

1. cell sample is applied to bank.

2. by using valve to connect magnetic post and flow control apparatus suitably.Magnetic field is put on pillar.Flow control apparatus allows the cell sample in the bank to pass through pillar with the low flow velocity of controlling.Cell with magnetic reaction is retained in the pillar.

3. randomly, damping fluid is applied to bank with flushing pillar (under magnetic field).

4. the change valve arrangement makes the magnetic post be connected with the buffer container fluid.

5. randomly, close magnetic field by pillar.

6. use the damping fluid flushing pillar that returns bank from buffer container with high flow rate.The non-specific cell that is retained in the pillar is washed back in the bank.

7. as required, repeatingstep 2 to 6.

8. with high flow rate the target cell wash-out is returned bank.

9. alternatively, remove pillar from magnetic field, and use the damping fluid flushing of introducing by bank from pillar wash-out target cell.

The magnetite gathering of embodiment 29 erythroblasts

The following example provides about the data from the enrichment of the nRBC of maternal blood.

Specimen preparation:

Collect 20mL blood separately in K from 8 different volunteer's donors₂In the EDTA anticoagulant tube, and in 6 hours, use the tripping device based on size as described herein to handle, wherein nucleated blood cell is collected in damping fluid (iDPBS, 1%BSA, 2mM EDTA).

The magnetite gathering step:

1. the cell sample with enrichment is applied to bank.

2. valve arrangement: the magnetic post is connected with flow control apparatus.The magnetic field of 1.4 teslas is put on pillar.Flow control apparatus allows the cell sample in the bank to pass through pillar with about 0.3mm/s.

3. the 3mL damping fluid is applied to bank with flushing pillar (under magnetic field).

4. the change valve arrangement makes the magnetic post be connected with the buffer container fluid.

5. use damping fluid to wash pillar, wash back bank with about 60mm/s from buffer container.

6. repeating step is 2 to 5 twice.

7. use the 3mL damping fluid target cell wash-out to be returned bank with 60mm/s.

8. alternatively, remove pillar from magnetic field, and use the damping fluid flushing of introducing by bank from pillar wash-out target cell.

The result

Enumerated the cell of the purifying of nRBC and WBC.Usually,＞99.95% WBC removes by this method, and nRBC then is resumed with the frequency of every mL whole blood greater than 2 nRBC in all situations.

Sample number	WBC number (10 in the whole blood6)	WBC counting (10 after the processing3/mL)	WBC removes	NRBC counts (nRBC/mL)
					1	185	99	99.95％	4.0
2	112	36	99.97％	5.4
					3	230	29	99.99％	2.9
4	275	101	99.96％	52.5
					5	222	52	99.98％	3.6
6	65	28	99.96％	59.0
					7	254	45	99.98％	5.9
8	122	31	99.97％	2.2

The productive rate ofembodiment 30 magnetite gatherings

The mixture that this embodiment uses white corpuscle (WBC) and red corpuscle (RBC) as sample characterization contain the productive rate of magnetic resolution method of the cell of oxyphorase.RBC is the target cell for the treatment of enrichment, and WBC is a cell to be removed.

Specimen preparation:

From 1 volunteer's donor with the 20mL blood collecting in K₂In the EDTA anticoagulant tube, and used the tripping device based on size as described herein to handle in 6 hours, wherein nucleated blood cell being divided a word with a hyphen at the end of a line with some RBC is collected in 3mL damping fluid (iDPBS, 1%BSA, 2mM EDTA).To sample characterization WBC counting (1,620 ten thousand) and RBC counting (7,600,000).

The magnetite gathering step:

1. cell sample is applied to bank.

2. valve arrangement: the magnetic post is connected with flow control apparatus.The magnetic field of 1.4 teslas is put on pillar.

Flow control apparatus allows the cell sample in the bank to pass through pillar with about 0.3mm/s.

3. the 3mL damping fluid is applied to bank with flushing pillar (under magnetic field).

4. the change valve arrangement makes the magnetic post be connected with the buffer container fluid.

5. use damping fluid to wash pillar, wash back bank with about 60mm/s from buffer container.

6. repeating step is 2 to 5 twice.

7. use the 3mL damping fluid target cell wash-out to be returned bank with 60mm/s.

The result

Enumerated the cell of the purifying of nRBC and WBC.The RBC counting is 7,500,000, and the WBC counting is 3200.Therefore, target cell (RBC) productive rate is removed greater than 98%, 99.98% non-target cell (WBC).

Embodiment 31 selective splittings

This embodiment provides a kind of selective splitting RBC that uses with the divide a word with a hyphen at the end of a line method of purifying erythroblast of low RBC.Selective splitting can be before magnetic resolution, in the process or carry out afterwards.

Step (selective splitting before the magnetic resolution):

1. from the karyocyte of whole blood or enrichment, use for example NH of chemical reagent₄Cl selective splitting erythroplastid;

2. at NaNO₂Handledkaryocyte 5 minutes in the (～50mM);

3. use magnetic resolution device as described herein to separate erythroblast and other karyocytes;

Step (selective splitting after the magnetic resolution):

1. from the karyocyte of whole blood or enrichment, in NaNO₂Handledkaryocyte 5 minutes in the (～50mM);

2. use magnetic resolution device as described herein to separate erythroblast and other karyocytes;

3. use for example NH of chemical reagent₄Cl selective splitting erythroplastid.

Step (selective splitting in the magnetic resolution process):

1. from the karyocyte of whole blood or enrichment, in NaNO₂Handledkaryocyte 5 minutes in the (～50mM);

2. use magnetic resolution device (the magnetic post wherein keeps nRBC) to separate erythroblast and other karyocytes.Make for example NH of chemical reagent₄Cl by pillar with the selective splitting erythroblast.By making inertia reagent stop scission reaction by pillar.

3. from pillar wash-out nRBC.

Cracking allows the scission reaction accurate timing in sepn process.

The cracking of embodiment 32 nucleus preparation

This embodiment provides a kind of magnetic enrichment and the nuclear method of full lysis from the blood separation erythroblast used.This method can comprise to be provided through the erythroblast sample of the enrichment of magnetic resolution preparation and makes this sample and chemical reagent for example acid alcohol (methyl alcohol and acetic acid 3: 1) or Triton solution (in iDPBS 0.2%).

Other embodiments

The application is incorporated in all publications, patent and the patent application mentioned in the above-mentioned specification sheets by reference into.Without departing from the scope and spirit of the present invention, the various modifications and variations of the method for the invention and system are apparent to those skilled in the art.Although be described in conjunction with a specific embodiment thereof the present invention, should understand the present invention who is advocated and should not be limited to these specific embodiments inadequately.The various modifications of the enforcement described mode of the present invention that in fact, it will be apparent to those skilled in the art are intended within the scope of the invention.

Other embodiments are in claims.

Claims (171)

Translated fromChinese

1.一种用于产生相对于第二组分富集了第一细胞或其组分的样品的装置，所述装置包括：1. A device for producing a sample enriched for a first cell or a component thereof relative to a second component, said device comprising:(a)通道，所述第一细胞或组分流经其；以及(a) a channel through which the first cell or component flows; and(b)磁体，其在所述通道中产生0.05忒斯拉至5.0忒斯拉的磁场和100忒斯拉/m至1,000,000忒斯拉/m的磁场梯度。(b) A magnet that produces a magnetic field of 0.05 Tesla to 5.0 Tesla and a magnetic field gradient of 100 Tesla/m to 1,000,000 Tesla/m in the channel.2.权利要求1的装置，其中所述第一细胞或组分保留在所述通道中，且所述第二组分不保留在所述通道中。2. The device of claim 1, wherein said first cell or component is retained in said channel and said second component is not retained in said channel.3.权利要求1的装置，其中所述第一细胞或组分不保留在所述通道中，且所述第二组分保留在所述通道中。3. The device of claim 1, wherein said first cell or component is not retained in said channel and said second component is retained in said channel.4.权利要求1的装置，其中所述通道包括第一出口和第二出口，且所述第一细胞或其组分被导入所述第一出口，而所述第二组分被导入所述第二出口。4. The device of claim 1, wherein said channel comprises a first outlet and a second outlet, and said first cells or components thereof are introduced into said first outlet and said second components are introduced into said Second exit.5.权利要求1的装置，其还包括分析组件，所述分析组件基于尺寸、形状、可变形性或亲和力来富集所述第一细胞或组分。5. The device of claim 1, further comprising an analysis component that enriches for the first cell or component based on size, shape, deformability, or affinity.6.权利要求5的装置，其中所述分析组件包括第一通道，所述第一通道包括这样一种结构，即以不平行于所述结构中平均流动方向的方向确定性偏转具有大于临界尺寸的流体力学尺寸的颗粒，其中所述颗粒为所述第一细胞或组分或所述第二组分。6. The apparatus of claim 5, wherein said analysis assembly comprises a first channel comprising a structure that deflects deterministically in a direction that is not parallel to the mean flow direction in said structure with a diameter greater than a critical dimension A particle of a hydrodynamic size, wherein said particle is said first cell or component or said second component.7.权利要求1的装置，其还包括能够改变所述样品的所述第一细胞或组分或第二组分的磁性的试剂。7. The device of claim 1, further comprising a reagent capable of altering the magnetic properties of said first cell or component or second component of said sample.8.权利要求7的装置，其中所述试剂改变所述第一细胞或组分或所述第二组分中存在的蛋白质的磁性。8. The device of claim 7, wherein said reagent alters the magnetic properties of proteins present in said first cell or fraction or said second fraction.9.权利要求8的装置，其中所述蛋白质包含铁。9. The device of claim 8, wherein said protein comprises iron.10.权利要求9的装置，其中所述蛋白质是胎儿血红蛋白、成人血红蛋白、高铁血红蛋白、肌红蛋白或细胞色素。10. The device of claim 9, wherein said protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.11.权利要求8的装置，其中所述试剂包括亚硝酸钠、二氧化碳、氧、一氧化碳或氮。11. The device of claim 8, wherein the reagent comprises sodium nitrite, carbon dioxide, oxygen, carbon monoxide, or nitrogen.12.权利要求1的装置，其中所述第一细胞是血细胞。12. The device of claim 1, wherein said first cell is a blood cell.13.权利要求1的装置，其中所述第一细胞是有核细胞。13. The device of claim 1, wherein said first cell is a nucleated cell.14.权利要求1的装置，其中所述第一细胞是无核细胞。14. The device of claim 1, wherein said first cell is an anucleated cell.15.权利要求1的装置，其中所述血细胞是成人有核红细胞。15. The device of claim 1, wherein said blood cells are adult nucleated red blood cells.16.权利要求1的装置，其中所述血细胞是胎儿有核红细胞。16. The device of claim 1, wherein said blood cells are fetal nucleated red blood cells.17.权利要求16的装置，其中所述胎儿有核红细胞来自少于10周龄的胎儿。17. The device of claim 16, wherein said fetal nucleated red blood cells are from a fetus less than 10 weeks old.18.权利要求1的装置，其中所述第一细胞是哺乳动物、鸟类、爬虫类动物或两栖类动物的。18. The device of claim 1, wherein said first cell is mammalian, avian, reptilian, or amphibian.19.权利要求1的装置，其中所述第一细胞的所述组分选自细胞核、核周区室、核膜、线粒体、叶绿体、或细胞膜、脂质、多糖、蛋白质、核酸、病毒颗粒以及核糖体。19. The device of claim 1, wherein said components of said first cell are selected from the group consisting of a nucleus, a perinuclear compartment, a nuclear membrane, mitochondria, chloroplasts, or cell membranes, lipids, polysaccharides, proteins, nucleic acids, viral particles, and Ribosome.20.权利要求7的装置，其中所述试剂导致在所述第一细胞或组分或所述第二组分中具有磁性的蛋白质表达或过表达。20. The device of claim 7, wherein said reagent results in expression or overexpression of a protein that is magnetic in said first cell or component or said second component.21.权利要求20的装置，其中所述试剂能够用具有磁反应性蛋白质转染所述第一细胞或所述第二组分。21. The device of claim 20, wherein said reagent is capable of transfecting said first cell or said second component with a magnetically responsive protein.22.权利要求7的装置，其中所述试剂包括结合至或掺入所述第一细胞或组分或所述第二组分的磁性颗粒。22. The device of claim 7, wherein said reagent comprises magnetic particles bound to or incorporated into said first cell or component or said second component.23.权利要求1的装置，其还包括泵，所述泵能够产生每秒钟大于50,000个细胞或其组分流入所述通道的流速。23. The device of claim 1, further comprising a pump capable of generating a flow rate of greater than 50,000 cells or components thereof per second into said channel.24.权利要求1的装置，其中至少90％的所述第一细胞或组分保留在所述装置中，且至少90％的所述第二组分不保留在所述装置中。24. The device of claim 1, wherein at least 90% of said first cells or components are retained in said device and at least 90% of said second components are not retained in said device.25.一种用于产生相对于第二组分富集了第一细胞或其组分的样品的方法，所述方法包括下列步骤：25. A method for producing a sample enriched in first cells or components thereof relative to a second component, said method comprising the steps of:(a)将包含所述第一细胞或组分的样品引入权利要求1的装置；(a) introducing a sample comprising said first cell or component into the device of claim 1;(b)使得所述样品中的所述第一细胞或组分或所述第二组分基于磁性而相对于另一组分被改变路径，从而产生富集了所述第一细胞或组分的所述样品。(b) causing said first cell or fraction or said second fraction in said sample to be rerouted relative to the other fraction based on magnetism, thereby producing enriched said first cell or fraction of the samples.26.权利要求25的方法，其中在步骤(a)中被引入所述装置的所述样品相对第三组分富集所述第一细胞或组分。26. The method of claim 25, wherein said sample introduced into said device in step (a) is enriched for said first cell or component relative to a third component.27.权利要求26的方法，其中在步骤(a)之前，使所述样品与分析组件接触，所述分析组件基于尺寸、形状、可变形性或亲和力相对于所述第三组分富集所述第一细胞或组分。27. The method of claim 26, wherein prior to step (a), the sample is contacted with an analysis component that is enriched for the third component based on size, shape, deformability, or affinity. the first cell or component.28.权利要求27的方法，其中所述分析组件包括第一通道，所述第一通道包括这样一种结构，即以不平行于所述结构中平均流动方向的方向确定性偏转具有大于临界尺寸的流体力学尺寸的颗粒，其中所述颗粒为所述样品的所述第一细胞或组分或所述第三组分。28. The method of claim 27, wherein said analysis assembly comprises a first channel comprising a structure that deflects deterministically in a direction non-parallel to the mean flow direction in said structure with a value greater than a critical dimension A particle of a hydrodynamic size, wherein said particle is said first cell or component or said third component of said sample.29.权利要求25的方法，其中富集了所述第一细胞或组分的所述样品保留至少70％的存在于所述样品中的所述第一细胞或组分。29. The method of claim 25, wherein said sample enriched for said first cell or component retains at least 70% of said first cell or component present in said sample.30.权利要求25的方法，其中富集了所述第一细胞或组分的所述样品被富集100倍。30. The method of claim 25, wherein said sample enriched for said first cell or component is enriched 100-fold.31.权利要求25的方法，其中富集了所述第一细胞的所述样品被富集至少1000倍，且所述第一细胞是有核红细胞。31. The method of claim 25, wherein said sample enriched for said first cell is at least 1000-fold enriched, and said first cell is a nucleated red blood cell.32.权利要求25的方法，其还包括在步骤(b)之前使所述样品与能够改变所述第一细胞或组分或第二组分的磁性的试剂接触的步骤。32. The method of claim 25, further comprising the step of contacting said sample with an agent capable of altering the magnetic properties of said first cell or component or second component prior to step (b).33.权利要求32的方法，其中所述试剂改变存在于所述第一细胞或组分或所述第二组分中的蛋白质的磁性。33. The method of claim 32, wherein said agent alters the magnetic properties of a protein present in said first cell or fraction or said second fraction.34.权利要求33的方法，其中所述蛋白质是胎儿血红蛋白、成人血红蛋白、高铁血红蛋白、肌红蛋白或细胞色素。34. The method of claim 33, wherein the protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.35.权利要求33的方法，其中所述试剂包括亚硝酸钠、二氧化碳、氧、一氧化碳或氮。35. The method of claim 33, wherein the reagent comprises sodium nitrite, carbon dioxide, oxygen, carbon monoxide, or nitrogen.36.权利要求25的方法，其中所述第一细胞是血细胞。36. The method of claim 25, wherein said first cell is a blood cell.37.权利要求25的方法，其中所述第一细胞是有核细胞。37. The method of claim 25, wherein said first cell is a nucleated cell.38.权利要求25的方法，其中所述第一细胞是无核细胞。38. The method of claim 25, wherein said first cell is an anucleated cell.39.权利要求36的方法，其中所述血细胞是成人有核红细胞。39. The method of claim 36, wherein said blood cells are adult nucleated red blood cells.40.权利要求39的方法，其中所述成人有核红细胞用于红白血病、β-重型地中海贫血、Bart氏血红蛋白或免疫性溶血性贫血的诊断或治疗监测。40. The method of claim 39, wherein said adult nucleated erythrocytes are used for diagnosis or treatment monitoring of erythroleukemia, beta-thalassemia major, Bart's hemoglobin, or immune hemolytic anemia.41.权利要求36的方法，其中所述血细胞是胎儿有核红细胞。41. The method of claim 36, wherein said blood cells are fetal nucleated red blood cells.42.权利要求41的方法，其中所述胎儿有核红细胞来自少于10周龄的胎儿。42. The method of claim 41, wherein said fetal nucleated red blood cells are from a fetus less than 10 weeks old.43.权利要求25的方法，其中所述第一细胞是哺乳动物、鸟类、爬虫类动物或两栖类动物的。43. The method of claim 25, wherein said first cell is mammalian, avian, reptilian, or amphibian.44.权利要求25的方法，其中所述第一细胞的所述组分选自细胞核、核周区室、核膜、线粒体、叶绿体、或细胞膜、脂质、多糖、蛋白质、核酸、病毒颗粒以及核糖体。44. The method of claim 25, wherein said component of said first cell is selected from the group consisting of nucleus, perinuclear compartment, nuclear envelope, mitochondria, chloroplast, or cell membrane, lipid, polysaccharide, protein, nucleic acid, viral particle, and Ribosome.45.权利要求32的方法，其中所述试剂导致在所述第一细胞或组分或所述第二组分中具有磁性的蛋白质表达或过表达。45. The method of claim 32, wherein said agent results in expression or overexpression of a protein that is magnetic in said first cell or fraction or said second fraction.46.权利要求32的方法，其中所述试剂包括结合至或掺入所述第一细胞或组分或所述第二组分的磁性颗粒。46. The method of claim 32, wherein said reagent comprises a magnetic particle bound to or incorporated into said first cell or component or said second component.47.权利要求25的方法，其中富集了所述第一细胞或组分的所述样品包括至少90％的在步骤(a)引入所述样品的所述第一细胞或组分，且包括少于10％的在步骤(b)引入所述样品的所述第二组分。47. The method of claim 25, wherein said sample enriched for said first cell or component comprises at least 90% of said first cell or component introduced into said sample in step (a), and comprises Less than 10% of said second component is introduced into said sample in step (b).48.权利要求25的方法，其中每秒钟大于50,000个细胞或其组分流入所述通道中。48. The method of claim 25, wherein greater than 50,000 cells or components thereof flow into said channel per second.49.一种用于产生相对于第二组分富集了第一细胞或其组分的样品的方法，所述方法包括下列步骤：49. A method for producing a sample enriched in a first cell or a component thereof relative to a second component, said method comprising the steps of:(a)使包含所述第一细胞或组分的样品与改变所述样品的所述第一细胞或组分或所述第二组分中表达的蛋白质的磁性的试剂接触，以产生改变的样品；(a) contacting a sample comprising said first cell or component with an agent that alters the magnetic properties of a protein expressed in said first cell or component or said second component of said sample to produce an altered sample;(b)使所述改变的样品与具有磁体的通道接触，所述磁体相对所述通道设置并产生磁场和磁场梯度，所述磁场和磁场梯度能够改变所述第一细胞或组分或所述第二组分相对另一组分的路径，从而产生富集了所述第一细胞或组分的所述样品。(b) contacting said altered sample with a channel having a magnet disposed relative to said channel and producing a magnetic field and magnetic field gradient capable of altering said first cell or component or said The pathway of a second component relative to another component, thereby producing said sample enriched for said first cell or component.50.权利要求49的方法，其中在步骤(a)之前或之后，包含所述第一细胞或组分的所述样品相对第三组分富集所述第一细胞或组分。50. The method of claim 49, wherein said sample comprising said first cell or fraction is enriched for said first cell or fraction relative to a third fraction before or after step (a).51.权利要求50的方法，其中在步骤(a)之前，使所述样品与分析组件接触，所述分析组件基于尺寸、形状、可变形性或亲和力而相对于所述第三组分富集所述第一细胞或组分。51. The method of claim 50, wherein prior to step (a), the sample is contacted with an analysis component that is enriched relative to the third component based on size, shape, deformability, or affinity said first cell or component.52.权利要求5 1的方法，其中所述分析组件包括第一通道，所述第一通道包括这样一种结构，即以不平行于所述结构中平均流动方向的方向确定性偏转具有大于临界尺寸的流体力学尺寸的颗粒，其中所述颗粒为所述第一细胞或组分或所述第二组分。52. The method of claim 51 , wherein said analysis assembly comprises a first channel comprising a structure that deterministically deflects in a direction that is not parallel to the mean flow direction in said structure with a value greater than a critical A hydrodynamically sized particle of the size wherein said particle is said first cell or component or said second component.53.权利要求49的方法，其中富集所述第一细胞或组分的所述样品保留至少70％的存在于所述样品中的所述第一细胞或组分。53. The method of claim 49, wherein said sample enriched for said first cell or component retains at least 70% of said first cell or component present in said sample.54.权利要求49的方法，其中富集所述第一细胞或组分的所述样品被富集100倍。54. The method of claim 49, wherein said sample enriched for said first cell or component is enriched 100-fold.55.权利要求49的方法，其中富集所述第一细胞或组分的所述样品被富集至少1000倍，且所述第一细胞是有核红细胞。55. The method of claim 49, wherein said sample enriched for said first cell or fraction is at least 1000-fold enriched and said first cell is a nucleated red blood cell.56.权利要求49的方法，其中所述试剂改变存在于所述第一细胞或组分或所述第二组分中的蛋白质的磁性。56. The method of claim 49, wherein said agent alters the magnetic properties of a protein present in said first cell or fraction or said second fraction.57.权利要求56的方法，其中所述蛋白质是胎儿血红蛋白、成人血红蛋白、高铁血红蛋白、肌红蛋白或细胞色素。57. The method of claim 56, wherein the protein is fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.58.权利要求56的方法，其中所述试剂包括亚硝酸钠、二氧化碳、氧、一氧化碳或氮。58. The method of claim 56, wherein the reagent comprises sodium nitrite, carbon dioxide, oxygen, carbon monoxide, or nitrogen.59.权利要求49的方法，其中所述第一细胞是血细胞。59. The method of claim 49, wherein said first cell is a blood cell.60.权利要求49的方法，其中所述第一细胞是有核细胞。60. The method of claim 49, wherein said first cell is a nucleated cell.61.权利要求49的方法，其中所述第一细胞是无核细胞。61. The method of claim 49, wherein said first cell is an anucleated cell.62.权利要求59的方法，其中所述血细胞是成人有核红细胞。62. The method of claim 59, wherein said blood cells are adult nucleated red blood cells.63.权利要求62的方法，其中所述成人有核红细胞用于红白血病、β-重型地中海贫血、Bart氏血红蛋白或免疫性溶血性贫血的诊断或治疗监测。63. The method of claim 62, wherein said adult human nucleated erythrocytes are used for diagnosis or treatment monitoring of erythroleukemia, beta-thalassemia major, Bart's hemoglobin, or immune hemolytic anemia.64.权利要求59的方法，其中所述血细胞是胎儿有核红细胞。64. The method of claim 59, wherein said blood cells are fetal nucleated red blood cells.65.权利要求64的方法，其中所述胎儿有核红细胞来自少于10周龄的胎儿。65. The method of claim 64, wherein said fetal nucleated red blood cells are from a fetus less than 10 weeks old.66.权利要求49的方法，其中所述第一细胞是哺乳动物、鸟类、爬虫类动物或两栖类动物的。66. The method of claim 49, wherein said first cell is mammalian, avian, reptilian, or amphibian.67.权利要求49的方法，其中所述第一细胞的所述组分选自细胞核、核周区室、核膜、线粒体、叶绿体、或细胞膜、脂质、多糖、蛋白质、核酸、病毒颗粒以及核糖体。67. The method of claim 49, wherein said component of said first cell is selected from the group consisting of nucleus, perinuclear compartment, nuclear envelope, mitochondria, chloroplast, or cell membrane, lipid, polysaccharide, protein, nucleic acid, viral particle, and Ribosome.68.权利要求49的方法，其中所述试剂导致在所述第一细胞或组分或所述第二组分中具有磁性的蛋白质表达或过表达。68. The method of claim 49, wherein said agent results in expression or overexpression of a protein that is magnetic in said first cell or fraction or said second fraction.69.权利要求49的方法，其中所述试剂包括结合至或掺入所述第一细胞或组分或所述第二组分的磁性颗粒。69. The method of claim 49, wherein said reagent comprises a magnetic particle bound to or incorporated into said first cell or component or said second component.70.权利要求49的方法，其中富集了所述第一细胞或组分的所述样品包括至少90％的在步骤(a)接触所述样品的所述第一细胞或组分，且包括少于10％的在步骤(b)接触所述样品的所述第二组分。70. The method of claim 49, wherein said sample enriched for said first cell or component comprises at least 90% of said first cell or component contacted with said sample in step (a), and comprises Less than 10% of said second component contacts said sample in step (b).71.权利要求49的方法，其中所述磁体在所述通道中产生0.05忒斯拉至5.0忒斯拉的磁场和100忒斯拉/m至1,000,000忒斯拉/m的磁场梯度。71. The method of claim 49, wherein said magnet produces a magnetic field of 0.05 Tesla to 5.0 Tesla and a magnetic field gradient of 100 Tesla/m to 1,000,000 Tesla/m in said channel.72.权利要求49的方法，其中每秒钟大于50,000个细胞或其组分流入所述通道中。72. The method of claim 49, wherein greater than 50,000 cells or components thereof flow into said channel per second.73.一种用于从含有第一分析物的流体样品相对于所述样品中的第二分析物和第三分析物富集所述第一分析物的方法，所述方法包括：73. A method for enriching a first analyte from a fluid sample containing the first analyte relative to a second analyte and a third analyte in the sample, the method comprising:(a)实施第一富集步骤，使用以第一方向指导所述第一分析物和以第二方向指导所述第二分析物的多个障碍物基于流体力学尺寸从所述流体样品富集所述第一分析物，以及(a) performing a first enrichment step enriching from the fluid sample based on hydrodynamic size using a plurality of obstacles directing the first analyte in a first direction and the second analyte in a second direction the first analyte, and(b)实施第二富集步骤，基于所述第一分析物或第三分析物的内在或外在磁性从所述流体样品富集所述第一分析物。(b) performing a second enrichment step of enriching said first analyte from said fluid sample based on intrinsic or extrinsic magnetic properties of said first or third analyte.74.权利要求73的方法，其中所述流体样品是血液样品。74. The method of claim 73, wherein said fluid sample is a blood sample.75.权利要求73的方法，其中所述流体样品是母体血液样品。75. The method of claim 73, wherein said fluid sample is a maternal blood sample.76.权利要求73的方法，其中所述一种或多种分析物是红细胞。76. The method of claim 73, wherein the one or more analytes are red blood cells.77.权利要求73的方法，其中所述一种或多种分析物是胎儿红细胞。77. The method of claim 73, wherein the one or more analytes are fetal red blood cells.78.权利要求73的方法，其中所述一种或多种分析物各自包括胎儿血红蛋白、成人血红蛋白、高铁血红蛋白、肌红蛋白或细胞色素。78. The method of claim 73, wherein each of the one or more analytes comprises fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.79.权利要求73的方法，其中所述第二富集步骤包括将磁场施加于所述第一富集步骤的产物。79. The method of claim 73, wherein said second enriching step comprises applying a magnetic field to the product of said first enriching step.80.权利要求79的方法，其中所述磁场吸引所述第一分析物或第三分析物。80. The method of claim 79, wherein the magnetic field attracts the first analyte or the third analyte.81.权利要求79的方法，其中所述磁场排斥所述第一分析物或第三分析物。81. The method of claim 79, wherein the magnetic field repels the first analyte or the third analyte.82.权利要求79的方法，其中所述磁场改变所述第一分析物相对于所述第三分析物的路径。82. The method of claim 79, wherein the magnetic field alters the path of the first analyte relative to the third analyte.83.权利要求79的方法，其中所述磁场在0.5忒斯拉和5.0忒斯拉之间。83. The method of claim 79, wherein said magnetic field is between 0.5 Tesla and 5.0 Tesla.84.权利要求79的方法，其中所述第二富集步骤还包括施加100忒斯拉/m至1,000,000忒斯拉/m的磁场梯度。84. The method of claim 79, wherein the second enriching step further comprises applying a magnetic field gradient of 100 Tesla/m to 1,000,000 Tesla/m.85.权利要求73的方法，其还包括对所述第一富集产物进行脱氧的步骤。85. The method of claim 73, further comprising the step of deoxygenating said first enriched product.86.权利要求85的方法，其中所述脱氧步骤包括使所述第一富集步骤的产物与CO、CO₂、N₂或NaNO₂接触。86. The method of claim 85, wherein said deoxygenating step comprises contacting the product of said first enrichment step with CO,_CO2 ,_N2, or_NaNO2 .87.权利要求73的方法，其还包括使所述第一分析物或第三分析物顺磁化的步骤。87. The method of claim 73, further comprising the step of paramagnetizing the first analyte or the third analyte.88.权利要求73的方法，其还包括使所述第一分析物或第三分析物去磁性的步骤。88. The method of claim 73, further comprising the step of demagnetizing the first analyte or the third analyte.89.权利要求73的方法，其中所述第一富集步骤和所述第二富集步骤顺序发生。89. The method of claim 73, wherein said first enrichment step and said second enrichment step occur sequentially.90.权利要求73的方法，其中所述第一富集步骤包括多个相互顺序发生的基于流体力学尺寸的富集步骤。90. The method of claim 73, wherein said first enrichment step comprises a plurality of mutually sequential hydrodynamic size-based enrichment steps.91.权利要求73的方法，其中所述第一富集步骤包括多个相互平行发生的基于流体力学尺寸的富集步骤。91. The method of claim 73, wherein said first enrichment step comprises a plurality of hydrodynamic size-based enrichment steps occurring in parallel to each other.92.权利要求73的方法，其中所述第二富集步骤包括多个相互平行发生的富集步骤。92. The method of claim 73, wherein said second enrichment step comprises a plurality of enrichment steps occurring in parallel with each other.93.权利要求73的方法，其中所述第一富集步骤在样品流经的过程中发生。93. The method of claim 73, wherein said first enrichment step occurs during flow through of the sample.94.权利要求73的方法，其中所述第二富集步骤在样品流经的过程中发生。94. The method of claim 73, wherein said second enrichment step occurs during flow through of the sample.95.权利要求73的方法，其中所述第二富集步骤基于内在磁性。95. The method of claim 73, wherein said second enrichment step is based on intrinsic magnetism.96.权利要求73的方法，其中所述第二富集步骤基于外在磁性。96. The method of claim 73, wherein said second enrichment step is based on extrinsic magnetism.97.权利要求73的方法，其中每秒钟大于50,000个分析物经受富集。97. The method of claim 73, wherein greater than 50,000 analytes are subjected to enrichment per second.98.一种系统，所述系统包括第一组件，所述第一组件具有：(a)以朝向第一出口的第一方向选择性指导具有大于临界尺寸的流体力学尺寸的一种或多种第一分析物和以朝向第二出口的第二方向选择性指导具有大于临界尺寸的流体力学尺寸的一种或多种第二分析物的障碍物阵列；(b)第二组件，其具有用于接收来自所述第一出口的所述一种或多种第一分析物的通道；以及(c)磁体，其在所述通道中产生改变所述一种或多种第一分析物路径的磁场和磁场梯度。98. A system comprising a first assembly having: (a) selectively directing in a first direction toward a first outlet one or more hydrodynamic dimensions greater than a critical dimension A first analyte and an array of obstacles that selectively direct one or more second analytes having a hydrodynamic dimension greater than the critical dimension in a second direction toward the second outlet; (b) a second assembly having a channel for receiving the one or more first analytes from the first outlet; and (c) a magnet that generates in the channel a change in the path of the one or more first analytes Magnetic fields and magnetic field gradients.99.权利要求98的系统，其中所述一种或多种第二分析物包括无核红细胞。99. The system of claim 98, wherein the one or more second analytes comprise anucleated red blood cells.100.权利要求98的系统，其中所述一种或多种第一分析物包括有核红细胞。100. The system of claim 98, wherein the one or more first analytes comprise nucleated red blood cells.101.权利要求98的系统，其中所述一种或多种第一分析物包括胎儿有核红细胞。101. The system of claim 98, wherein the one or more first analytes comprise fetal nucleated red blood cells.102.权利要求98的系统，其中所述一种或多种第一分析物包括胎儿血红蛋白、成人血红蛋白、高铁血红蛋白、肌红蛋白或细胞色素。102. The system of claim 98, wherein the one or more first analytes comprise fetal hemoglobin, adult hemoglobin, methemoglobin, myoglobin, or cytochrome.103.权利要求98的系统，其还包括与所述障碍物阵列或所述通道偶联的储库，所述储库包含脱氧剂。103. The system of claim 98, further comprising a reservoir coupled to the barrier array or the channel, the reservoir comprising a deoxidizer.104.权利要求98的系统，其还包括储库，所述储库包含用于特异性结合所述一种或多种第一分析物或其组分的探针。104. The system of claim 98, further comprising a reservoir comprising probes for specifically binding the one or more first analytes or components thereof.105.权利要求104的系统，其中所述探针是核酸探针或抗体探针。105. The system of claim 104, wherein the probe is a nucleic acid probe or an antibody probe.106.权利要求98的系统，其中所述磁场在0.5忒斯拉和5.0忒斯拉之间。106. The system of claim 98, wherein said magnetic field is between 0.5 Tesla and 5.0 Tesla.107.权利要求98的系统，其中所述磁场梯度在100忒斯拉/m和1,000,000忒斯拉/m之间。107. The system of claim 98, wherein the magnetic field gradient is between 100 Tesla/m and 1,000,000 Tesla/m.108.权利要求98的系统，其中所述一种或多种第一分析物的路径基于内在磁性而被改变。108. The system of claim 98, wherein the path of the one or more first analytes is altered based on intrinsic magnetism.109.一种系统，所述系统包括：(a)具有二维障碍物阵列的流通通道，所述障碍物阵列以朝向第一出口的第一方向选择性指导具有大于临界尺寸的流体力学尺寸的一种或多种第一分析物和以朝向第二出口的第二方向选择性指导具有大于临界尺寸的流体力学尺寸的一种或多种第二分析物；以及(b)磁体，所述磁体产生改变所述一种或多种第一分析物路径的磁场和磁场梯度。109. A system comprising: (a) a flow channel having a two-dimensional array of obstacles that selectively directs a fluid having a hydrodynamic dimension greater than a critical dimension in a first direction toward a first outlet one or more first analytes and one or more second analytes selectively directing in a second direction toward the second outlet one or more second analytes having a hydrodynamic size greater than the critical size; and (b) a magnet that A magnetic field and a magnetic field gradient are generated that alter the path of the one or more first analytes.110.权利要求109的系统，其中所述一种或多种第二分析物包括胎儿有核红细胞。110. The system of claim 109, wherein the one or more second analytes comprise fetal nucleated red blood cells.111.权利要求109的系统，其中所述一种或多种第一分析物的路径基于血红蛋白的存在而被改变。111. The system of claim 109, wherein the pathway of the one or more first analytes is altered based on the presence of hemoglobin.112.权利要求109的系统，其中所述磁场在0.5忒斯拉和5.0忒斯拉之间。112. The system of claim 109, wherein said magnetic field is between 0.5 Tesla and 5.0 Tesla.113.权利要求109的系统，其中所述磁场梯度在100忒斯拉/m和1,000,000忒斯拉/m之间。113. The system of claim 109, wherein the magnetic field gradient is between 100 Tesla/m and 1,000,000 Tesla/m.114.一种用于产生富集了分析物的样品的装置，所述装置包括：114. A device for producing an analyte-enriched sample, the device comprising:(a)第一通道，所述第一通道包括下述结构：以不平行于所述结构的平均流动方向的方向确定性偏转流体力学尺寸大于临界尺寸的颗粒的结构，其中所述颗粒是分析物颗粒或是所述样品的非分析物组分；以及(a) a first channel comprising a structure that deterministically deflects particles of a hydrodynamic size greater than a critical size in a direction non-parallel to the mean flow direction of the structure, wherein the particles are analyzed particles or non-analyte components of the sample; and(b)储库，所述储库流体偶联至所述第一通道的出口，所述分析物通过所述出口进入所述储库，其中所述储库包括改变所述分析物磁性的试剂。(b) a reservoir fluidly coupled to an outlet of the first channel through which the analyte enters the reservoir, wherein the reservoir includes a reagent that alters the magnetic properties of the analyte .115.权利要求114的装置，其中所述第一通道是微流体通道。115. The device of claim 114, wherein said first channel is a microfluidic channel.116.权利要求114的装置，其中所述结构包括形成间隙网络的障碍物阵列，其中通过所述间隙的流体被不相等地分成主流和次流，使得所述主流的平均方向不平行于所述通道中的流体的平均方向。116. The device of claim 114, wherein said structure includes an array of obstacles forming a network of gaps, wherein fluid passing through said gaps is unequally divided into primary and secondary flows such that the average direction of said primary flow is not parallel to said The average direction of the fluid in the channel.117.权利要求116的装置，其中所述障碍物阵列包括第一排和第二排，其中所述第二排侧对所述第一排放置，使得流过所述第一排间隙的流体不相等地分入所述第二排的两个间隙。117. The apparatus of claim 116, wherein said array of obstacles comprises a first row and a second row, wherein said second row is positioned sideways to said first row such that fluid flowing through said first row of gaps does not Divide equally into the two gaps of the second row.118.权利要求114的装置，其中所述分析物具有大于所述临界尺寸的流体力学尺寸。118. The device of claim 114, wherein said analyte has a hydrodynamic size greater than said critical size.119.权利要求114的装置，其中所述分析物具有小于所述临界尺寸的流体力学尺寸。119. The device of claim 114, wherein said analyte has a hydrodynamic size smaller than said critical size.120.权利要求114的装置，其还包括能够产生磁场的磁体。120. The device of claim 114, further comprising a magnet capable of generating a magnetic field.121.权利要求120的装置，其中所述磁体包括配置在第二通道中的磁性障碍物的区域。121. The apparatus of claim 120, wherein the magnet comprises a region of magnetic barriers disposed in the second channel.122.权利要求121的装置，其中至少一部分所述磁性障碍物包括永久性磁体。122. The apparatus of claim 121, wherein at least a portion of said magnetic barriers comprise permanent magnets.123.权利要求121的装置，其中至少一部分所述磁性障碍物包括非永久性磁体。123. The apparatus of claim 121, wherein at least a portion of said magnetic barriers comprise non-permanent magnets.124.权利要求121的装置，其中所述障碍物以二维阵列排序。124. The apparatus of claim 121, wherein said obstacles are ordered in a two-dimensional array.125.权利要求121的装置，其中所述第二通道是微流体通道。125. The device of claim 121, wherein said second channel is a microfluidic channel.126.权利要求114的装置，其中所述储库还包括包含磁体的第二通道。126. The device of claim 114, wherein said reservoir further comprises a second channel comprising a magnet.127.权利要求114的装置，其中所述试剂改变所述一种或多种分析物的内在磁性。127. The device of claim 114, wherein said reagent alters the intrinsic magnetic properties of said one or more analytes.128.权利要求127的装置，其中所述试剂包括亚硝酸钠。128. The device of claim 127, wherein said reagent comprises sodium nitrite.129.权利要求114的装置，其中所述试剂结合所述一种或多种分析物。129. The device of claim 114, wherein said reagent binds said one or more analytes.130.权利要求129的装置，其中所述试剂包括磁性颗粒。130. The device of claim 129, wherein the reagent comprises magnetic particles.131.权利要求130的装置，其中所述磁性颗粒包括抗体或其抗原结合片段。131. The device of claim 130, wherein the magnetic particles comprise antibodies or antigen-binding fragments thereof.132.权利要求131的装置，其中所述抗体是抗CD71、抗CD36、抗CD45、抗GPA、抗抗原i、抗CD34或抗胎儿血红蛋白的抗体。132. The device of claim 131, wherein the antibody is an anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, or anti-fetal hemoglobin antibody.133.权利要求129的装置，其中所述试剂包括全转铁蛋白。133. The device of claim 129, wherein said reagent comprises holotransferrin.134.一种用于产生相对于第二分析物富集了第一分析物的样品的方法，所述方法包括：134. A method for producing a sample enriched for a first analyte relative to a second analyte, the method comprising:(a)将至少一部分所述样品应用于包括下述结构的装置：以不平行于所述结构的平均流动方向的方向确定性偏转具有大于临界尺寸的流体力学尺寸的颗粒，从而产生富集了第一分析物并包括所述第二分析物的第二样品；(a) applying at least a portion of said sample to a device comprising a structure that deterministically deflects particles having a hydrodynamic size greater than a critical size in a direction non-parallel to the mean flow direction of said structure, thereby producing an enriched a first analyte and a second sample comprising said second analyte;(b)使所述第二样品与改变所述第一分析物磁性的试剂结合以产生改变的第一分析物；以及(b) binding the second sample to an agent that alters the magnetic properties of the first analyte to produce an altered first analyte; and(c)将磁场施加于所述第二样品，其中所述磁场产生差异力来物理分离所述改变的第一分析物和所述第二分析物，从而产生富集了所述第一分析物的样品。(c) applying a magnetic field to said second sample, wherein said magnetic field creates a differential force to physically separate said altered first analyte and said second analyte, thereby producing a magnetic field enriched for said first analyte. sample.135.权利要求134的方法，其中所述试剂结合所述第一分析物。135. The method of claim 134, wherein said reagent binds said first analyte.136.权利要求134的方法，其中所述试剂改变所述第一分析物的内在磁性。136. The method of claim 134, wherein said reagent alters the intrinsic magnetic properties of said first analyte.137.权利要求136的方法，其中所述试剂包括亚硝酸钠。137. The method of claim 136, wherein the reagent comprises sodium nitrite.138.权利要求134的方法，其中所述试剂包括结合至或掺入所述第一分析物的磁性颗粒。138. The method of claim 134, wherein said reagent comprises magnetic particles that bind to or incorporate said first analyte.139.权利要求138的方法，其中所述磁性颗粒包括抗体或其抗原结合片段。139. The method of claim 138, wherein the magnetic particles comprise antibodies or antigen-binding fragments thereof.140.权利要求139的方法，其中所述抗体是抗CD71、抗CD36、抗CD45、抗GPA、抗抗原i、抗CD34或抗胎儿血红蛋白的抗体。140. The method of claim 139, wherein the antibody is an anti-CD71, anti-CD36, anti-CD45, anti-GPA, anti-antigen i, anti-CD34, or anti-fetal hemoglobin antibody.141.权利要求134的方法，其中所述分析物具有大于所述临界尺寸的流体力学尺寸。141. The method of claim 134, wherein said analyte has a hydrodynamic size greater than said critical size.142.权利要求134的方法，其中所述分析物具有小于所述临界尺寸的流体力学尺寸。142. The method of claim 134, wherein said analyte has a hydrodynamic size smaller than said critical size.143.权利要求134的方法，其中所述样品包括母体血液样品。143. The method of claim 134, wherein said sample comprises a maternal blood sample.144.权利要求134的方法，其中所述第一分析物是细胞、细胞器或病毒。144. The method of claim 134, wherein the first analyte is a cell, organelle, or virus.145.权利要求144的方法，其中所述细胞是细菌细胞、胎儿细胞或血液细胞。145. The method of claim 144, wherein the cells are bacterial cells, fetal cells, or blood cells.146.权利要求145的方法，其中所述血液细胞是胎儿红细胞。146. The method of claim 145, wherein said blood cells are fetal red blood cells.147.权利要求144的方法，其中所述细胞器是细胞核。147. The method of claim 144, wherein the organelle is a nucleus.148.一种产生相对于第二血液组分富集了红细胞的样品的方法，所述方法包括：148. A method of producing a sample enriched in red blood cells relative to a second blood component, the method comprising:(a)使包括红细胞的样品与使铁氧化的试剂接触而产生氧化血红蛋白；以及(a) producing oxyhemoglobin by contacting a sample comprising red blood cells with an agent that oxidizes iron; and(b)将磁场施加于所述样品，其中具有氧化血红蛋白的所述红细胞以大于所述第二血液组分的程度被吸引至所述磁场，从而产生富集了所述红细胞的所述样品。(b) applying a magnetic field to the sample, wherein the red blood cells having oxidized hemoglobin are attracted to the magnetic field to a greater extent than the second blood component, thereby producing the sample enriched in the red blood cells.149.权利要求148的方法，其中所述红细胞是胎儿红细胞。149. The method of claim 148, wherein said red blood cells are fetal red blood cells.150.权利要求149的方法，其中所述第二血液组分是母体血细胞。150. The method of claim 149, wherein said second blood component is maternal blood cells.151.权利要求148的方法，其中在所述步骤(a)之前，富集所述样品的所述红细胞。151. The method of claim 148, wherein said red blood cells of said sample are enriched prior to said step (a).152.权利要求151的方法，其中所述富集是通过将至少一部分所述样品应用于包括下述结构的装置而进行，即以不平行于所述结构的平均流动方向的方向确定性偏转流体力学尺寸大于临界尺寸的颗粒的结构。152. The method of claim 151, wherein said enriching is performed by applying at least a portion of said sample to a device comprising a structure that deterministically deflects fluid in a direction that is not parallel to the mean flow direction of said structure The structure of particles whose mechanical size is larger than the critical size.153.权利要求152的方法，其中所述胎儿红细胞相对于所述母体红细胞而被富集。153. The method of claim 152, wherein said fetal red blood cells are enriched relative to said maternal red blood cells.154.一种用于产生富集了红细胞的样品的装置，所述装置包括：154. A device for producing a sample enriched in red blood cells, the device comprising:(a)分析装置，其基于尺寸、形状、可变形性或亲和力富集所述红细胞的；以及(a) an assay device that enriches said red blood cells based on size, shape, deformability or affinity; and(b)储库，其包含可氧化铁的试剂，其中所述试剂增加所述红细胞的磁反应性。(b) a reservoir comprising an iron oxidizable reagent, wherein said reagent increases the magnetic reactivity of said red blood cells.155.权利要求154的装置，其中所述分析装置包括包含下述结构的第一通道，即以不平行于所述结构的平均流动方向的方向确定性偏转流体力学尺寸大于临界尺寸的颗粒的结构。155. The device of claim 154, wherein said analysis device comprises a first channel comprising a structure that deterministically deflects particles of a hydrodynamic size greater than a critical size in a direction non-parallel to the mean flow direction of said structure .156.权利要求154的装置，其中所述试剂是亚硝酸钠。156. The device of claim 154, wherein the reagent is sodium nitrite.157.一种用于富集红系细胞亚群的方法，所述方法包括下列步骤：157. A method for enriching a subpopulation of erythroid cells, said method comprising the steps of:(a)将包括红系细胞的样品引入柱子中，并将磁场施加于所述柱子；以及(a) introducing a sample comprising erythroid cells into a column, and applying a magnetic field to said column; and(b)使所述样品中的第一类型红系细胞的路径相对于第二类型红系细胞基于磁性而改变，其中所述第一类型红系细胞受所述磁场的吸引大于所述第二类型，从而富集红系细胞亚群。(b) altering the path of erythroid cells of a first type in the sample relative to erythroid cells of a second type based on magnetism, wherein the erythroid cells of the first type are more attracted to the magnetic field than the second type of erythroid cells type, thereby enriching erythroid cell subsets.158.权利要求157的方法，其中所述第一类型红系细胞是正染性正成红细胞。158. The method of claim 157, wherein said first type of erythroid cells are orthochromatic erythroblasts.159.权利要求157的方法，其中所述第一类型红系细胞是成熟红细胞。159. The method of claim 157, wherein said first type of erythroid cells are mature erythrocytes.160.权利要求157的方法，其中所述第二类型红系细胞是多染性正成红细胞。160. The method of claim 157, wherein said second type of erythroid cells are polychromatic normal erythroblasts.161.权利要求157的方法，其中所述第二类型红系细胞是正染性正成红细胞。161. The method of claim 157, wherein said second type of erythroid cell is an orthochromatic erythroblast.162.一种用于富集具有纳入的磁性易感颗粒的细胞群的方法，所述方法包括下列步骤：162. A method for enriching a population of cells with incorporated magnetically susceptible particles, said method comprising the steps of:(a)将包括红细胞的样品引入柱子中，并将磁场施加于所述柱子；以及(a) introducing a sample comprising red blood cells into a column, and applying a magnetic field to said column; and(b)使所述样品中具有纳入的磁易感颗粒的细胞的路径相对于所述样品中第二类型细胞基于磁性而改变，从而富集所述细胞群。(b) magnetically altering the path of cells having incorporated magnetically susceptible particles in the sample relative to cells of a second type in the sample, thereby enriching the population of cells.163.权利要求162的方法，其中所述磁易感颗粒是红细胞。163. The method of claim 162, wherein said magnetically susceptible particles are red blood cells.164.权利要求162的方法，其中所述磁易感颗粒是趋磁细菌。164. The method of claim 162, wherein said magnetically susceptible particle is a magnetotactic bacterium.165.权利要求162的方法，其中所述磁易感颗粒包括磁铁石或硫铁石。165. The method of claim 162, wherein said magnetically susceptible particles comprise magnetite or pyrite.166.权利要求162的方法，其中具有纳入的磁易感颗粒的所述细胞为单核细胞或巨噬细胞。166. The method of claim 162, wherein said cells having incorporated magnetically susceptible particles are monocytes or macrophages.167.权利要求162的方法，其中具有纳入的磁易感颗粒的所述细胞用于家族性噬血细胞性组织细胞增多症、急性单核细胞性白血病或淋巴瘤的诊断或治疗监测。167. The method of claim 162, wherein said cells with incorporated magnetically susceptible particles are used for diagnosis or treatment monitoring of familial hemophagocytic histiocytosis, acute monocytic leukemia, or lymphoma.168.一种用于清除血液样品的红细胞和白细胞的方法，所述方法包括下列步骤：168. A method for depleting a blood sample of red blood cells and white blood cells, said method comprising the steps of:(a)将包括红细胞的样品引入柱子中，并将磁场施加于所述柱子；(a) introducing a sample comprising red blood cells into a column, and applying a magnetic field to said column;(b)在步骤(a)之前、过程中或之后，使所述样品与结合白细胞的磁易感试剂接触；以及(b) contacting said sample with a leukocyte-binding magnetic susceptibility reagent before, during or after step (a); and(c)使所述样品中红细胞和白细胞的路径相对于第三类型细胞基于磁性而改变，从而清除所述样品的红细胞和白细胞。(c) changing the path of red blood cells and white blood cells in the sample relative to a third type of cells based on magnetism, thereby depleting the sample of red blood cells and white blood cells.169.权利要求168的方法，其中所述第三类型细胞包括干细胞。169. The method of claim 168, wherein said third type of cells comprises stem cells.170.权利要求168的方法，其中所述血液样品是脐带血液样品。170. The method of claim 168, wherein said blood sample is an umbilical cord blood sample.171.权利要求168的方法，其中所述磁易感试剂包括抗CD45或抗CD15抗体。171. The method of claim 168, wherein said magnetic susceptibility agent comprises an anti-CD45 or anti-CD15 antibody.

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