HK1116720B - Cell separation using microchannel having patterned posts - Google Patents

Description

Cell separation using microchannels with patterned posts

The present invention relates to the extraction or isolation of target biomolecules from a feed liquid, and more particularly to a method and apparatus for isolating desired target human cells from a body fluid or the like.

Background

Efficient isolation and collection of rare cells from heterogeneous cell populations remains of great interest because of the growing need to isolate cell populations for use in disease diagnosis and therapy (e.g., gene therapy) and basic scientific research. For example, diseased cells (e.g., cancer cells) in a larger normal cell population can be isolated and the depleted cell population can be returned to the patient.

Cell isolation is a rapidly growing area in biomedical and clinical development, and improvements in methods for isolating desired subsets of cells from complex cell populations have broadened the search and application for relatively homogeneous and well-defined cells. Isolated cells are also widely used in research, such as determining the effect of drugs or therapies on target cell populations, studying biological pathways, isolating and studying transformed or modified cell populations, and the like. Current clinical applications include, for example, the isolation of hematopoietic stem cells for the reconstitution of blood cells, particularly in combination with invasive chemotherapy and radiation therapy.

Published U.S. patent application No.2004/038315 describes attaching a releasable linker to the surface of the lumen of a capillary vessel to bind desired cells, followed by release and recovery of the cells by a cleavage reagent. Published U.S. patent application No.2002/132316 utilizes a microchannel device to separate cell populations by employing a moving gradient light field. U.S. Pat. No.6,074,827 discloses an "enrichment channel" constructed using a microfluidic (bed) device, in which electrophoresis is used to separate and identify specific nucleic acids in a sample. It is also mentioned that antibodies or other binding fragments may optionally be utilized to retain the desired target biological substance. U.S. patent No.6,432,630 discloses the use of a microfluidic system for directing a biological particle-containing liquid through a channel to be selectively biased (collected), indicating that the system can be used to separate fetal cells from a maternal blood sample.

U.S. Pat. No.6,454,924 discloses microfluidic devices that allow a liquid sample containing an analyte to flow across a surface on which upstanding pillars are disposed, the pillars having capture reagents bound thereto, the sides of the pillars being hydrophobic to facilitate restricting the flow of liquid in the channels.

K.takahashi et al, j.nanobiotechnology.2, 5: 6(2004, 6, 13) discloses an on-chip cell sorting system in which a plurality of microfluidic inlet channels lead to a central cell sorting region formed in a PDMS plate (made of photoresist epoxy using a master mold) mounted on a glass plate. Agar gel electrodes in the PDMS plate facilitate the separation (removal) of unwanted cells by applying electrostatic forces, which are then forced into a parallel continuous flow of waste buffer as it flows through the short cell sorting zone, which is convergent. It has also been shown that large dust particles can be physically trapped using pre-filtration. Published U.S. patent application No.2004/029221 discloses a similar structured microfluidic device that can be used for cell separation, such as separation of fetal cells in maternal blood by selective lysis of maternal RBCs (red blood cells). Whole blood can be separated by introducing a sample containing cells into a microfluidic channel device that contains multiple barriers with appropriately coupled binding molecules (e.g., antibodies) on the surfaces that bind to the cells in the sample. U.S. patent No.5,637,469 discloses a microfluidic device containing multiple channels 100 microns deep or less with binding molecules (e.g., antibodies) immobilized on the surface to capture biomolecules of interest for in situ analysis. U.S. Pat. No.5,147,607 describes a device for immobilizing antibodies in microchannels for immunoassays, such as sandwich assays. The recessed region in the microchannel may contain a set of protrusions extending upwardly from the bottom surface of the channel, to which the antibody is immobilized.

The above briefly mentioned references show that there is still a constant search for improved methods for separating cells or other biological material in body fluids and the like.

Summary of The Invention

The present invention provides a microfluidic device that can be used to recover rare cells or other target biomolecules in a smaller volume of bodily fluid or the like, or in which at least one specially constructed microchannel device can be disposed. The device is constructed to form a channel-like flow path (hereinafter referred to as a flow path) in the substrate used, and a plurality of posts are laterally fixed in the collection region. The posts are integral with the substrate and extend between the upper and lower surfaces of the channel. The posts are arranged in a specific irregular array pattern to disrupt straight-line flow therethrough, and it is critical to disrupt regular streamlined flow through the array to ensure that bodily or other liquids flowing along this flow path into the collection region collide with the posts and promote their rotation and turbulence. The posts vary in size (e.g., cross-sectional diameter). The chelating agent is selected to appropriately bind to the surfaces of the transverse posts and all other surfaces throughout the collection region to capture the desired target molecules, which are collected in the collection region of the microchannel. It is preferred to place the supply well upstream of the microchannel leading to the collection region. It was found that when the microfluidic device was oriented obliquely to the horizontal, more complete cell separation was obtained due to the force vector effect created by gravity.

In a particular aspect, the invention provides a method of separating biomolecules (e.g. cells) from a bodily fluid or other liquid sample, the method comprising flowing the sample containing target biomolecules downstream from an inlet to an outlet along a flow path of a microfluidic device, said flow path comprising a microchannel arrangement including a collection region of enlarged cross-section, and the device being oriented such that said flow path collection region is at an angle of about 30 to 60 degrees to the horizontal, separating the target biomolecules from the flowing sample by: (a) disrupting the straight-line flow through the collection region as a result of a plurality of separation posts disposed within the region blocking such flow, the posts are integral with the upper or lower surface of the microchannel and extend to the opposite side surface thereof, said posts extending transversely to said flow path and being arranged in an irregular pattern which extends laterally across said collection region and prevents linear and streamlined flow in the flow path, said collection region containing said posts carrying on all surfaces thereof a chelating agent, and (b) destructive effects on fluid flow due to force vectors generated by said irregular posts and gravity that form acute angles with said lower surface of said collection region, capturing target molecules in the flowing liquid sample at the collection region surface by binding the target molecules to the chelating agent, and expelling the remainder of the liquid sample through the outlet.

In another particular aspect, the invention provides a device for separating biomolecules (e.g. cells) from a bodily fluid or other liquid sample, the device comprising a body having a flow path therein through which a sample containing target biomolecules flows, and a sealing plate, the body having an inlet passage leading to the flow path, an outlet passage leading therefrom, and a microchannel arrangement extending between the inlet and outlet passages, the microchannel arrangement comprising a collection region having upper and lower surfaces, one of which is provided by the sealing plate, and a plurality of transverse separation posts, the posts being integral with one of the upper and lower surfaces of the collection region and extending laterally across the flow path to the other side surface provided by the sealing plate, the posts being arranged in an irregular pattern to disrupt the linear and streamlined flow through the region, the surfaces of the collection region, comprising said posts with a chelating agent to be bound to target biomolecules and said inlet being at an angle of about 120-150 degrees to said flow path through said collection region so that a sample can be introduced substantially vertically through said inlet and downwardly, and said body being arranged so that said flow path is at an angle of about 30-60 degrees to the horizontal so that the irregular pattern of said posts and the force vector created by gravity effectively trap target biomolecules at said collection region, especially the lower surface thereof.

In another particular aspect, the present invention provides a microfluidic device for separating biomolecules (e.g., cells) from a bodily fluid or other liquid sample, the device comprising: a body having a flow path therein defining a cavity in a planar surface through which a sample containing target biomolecules flows, the flow path having inlet means, outlet means and a microchannel arrangement extending between said inlet and outlet means, said microchannel arrangement comprising a collection region having a plurality of transverse discrete posts therein, and a sealing plate means having a planar surface adjacent the planar surface of the body and adjacent the flow path cavity, said posts being integral with and extending from the bottom surface of the collection region to the surface of the sealing plate means, said posts being arranged in an irregular pattern and extending laterally across the flow path within the collection region to disrupt straight and streamlined flow through the region, and all surfaces of the collection region comprising said posts coated with a permeable hydrophilic hydrogel and carrying a chelator to which target biomolecules are to be bound, whereby the irregular pattern of pillars disrupts streamlined flow through the collection region to effectively capture target biomolecules on the surface of the collection region.

Brief Description of Drawings

FIG. 1 is a perspective view of a substrate for a microfluidic device showing a simplified post-containing collection region fabricated in a microfluidic channel.

FIG. 2 is an enlarged fragmentary view showing the patterned posts located in the collection region portion of FIG. 1.

FIG. 3 is a front view of the substrate of FIG. 1 taken in cross-section along line 3-3 with a cover plate attached to its bottom surface.

FIG. 4 is a schematic perspective view of the substrate generally shown in FIG. 1 including two valves, with an intermediate plate.

Fig. 5 is a cross-sectional view taken along line 5-5 of fig. 4.

FIG. 6 is a schematic plan view of a substrate of the type shown in FIG. 1, wherein the pump fabricated is part of the fluidic device.

FIG. 7 is a schematic illustration of the portion of the substrate in the supply area to which the micro-mixer is added.

FIG. 8 is a representative illustration of the binding of antibodies throughout a collection region by the addition of a hydrophilic coating.

FIGS. 9 and 10 are representative illustrations of the chemistry used to bind chelators (e.g., antibodies) throughout the collection region using a hydrophilic coating, and depict the capture of desired target cells.

FIG. 11 is a flowchart illustrating the procedure of recovering cells using the pattern pillars, and a cell apparatus.

FIG. 12 is a perspective view of another embodiment of a microfluidic device designed to operate with the collection region of a microchannel device containing pillars inclined from the horizontal.

Fig. 13 is a bottom view of the microfluidic device body of fig. 12.

Fig. 14 is a front elevational view, with dimensions exaggerated, of the cross-section taken along line 14-14 of fig. 12.

Fig. 15 is a schematic view showing the device of fig. 12 in use.

Detailed description of the preferred embodiments

The device provided by the present invention comprises essentially a body or substrate 11 in which a defined flow-through pathway (hereinafter flow path) comprising at least one fluid channel 13 including a collection region 17 is defined, the flow path being connected to a sample inlet 15 and a liquid outlet 19. As described herein below, the flow path includes several microchannels arranged in series, each with a collection region. Alternatively, a microchannel may have more than one collection region arranged in series, and multiple inlets and multiple outlets, as is well known in the art. However, the body may be part of an integrated microfluidic (bed) device built on a chip, a disk or the like; in such devices, essentially the entire MEMS (micro-electro-mechanical system) or module required for cell recovery and/or biomolecule diagnostics in isolated samples can be incorporated as a compact, easily handled unit.

Fig. 1 is a perspective view of a body or substrate 11 in which a flow path is formed comprising a microchannel 13 to which a liquid sample is supplied through an opening or well 15 as an inlet and an opening 19 as an outlet. The cross-section of the collecting zone 17 is larger than the cross-section of the entry zone 18 leading to the inlet 15. The inlet zone has a pair of axially aligned splitter/support struts 21 located at the end of the inlet zone 18 upstream of the widened collection zone 17. These centrally located flow-splitting columns split the flow into two flow paths, which serve to more evenly distribute the flow of liquid to the entrance of the collection region 17. Within the collection region 17 are a plurality of upstanding posts 23 which are transversely disposed in the liquid flow path and are irregularly, generally randomly, disposed across the entire depth of the collection region portion of the fluid passageway. The posts are arranged so that the fluid flows through the collection region in a non-linear manner, thereby disrupting the streamlined flow and ensuring good contact between the liquid flowing along the flow path and the surfaces of the posts. The posts are integral with and extend perpendicularly from the collection region planar bottom surface 22, creating a surface perpendicular to the horizontal flow path of the liquid, directing the liquid through the fluid channels in the substrate 11. Preferably, the liquid extends across the facing surface of the bonded closure plate 27 to adhere to the free surface thereof, as described in more detail below, the closure plate 27 being parallel to the bottom surface 22 and acting to close the fluid passageway. The inlet and outlet holes 24a and 24b may be drilled in the closure plate, but are preferably formed in the substrate 11. Another liquid distribution/support column 21a is located at the outlet of the collection zone.

As is well known in the art, a substrate can be formed to include a pair of parallel microchannel flow paths each containing a collection region. Such flow paths may be used for continuous liquid flow, or may be used for parallel flow operations. The liquid flow may be pushed by a pump, such as a syringe pump or the like, or the liquid of the reservoir may be sucked through the large-diameter inlet hole 24a by a vacuum pump. Preferably, such wells are capable of holding about 50-500 microliters of liquid sample.

The fluid passageways are designed to provide a flow rate through the device within a reasonable range, such as about 0.05-5 mm/sec for maternal blood injection using a standard Harward syringe pump, in the collection region, which substantially disrupts streamlined flow through the region without turbulence, due to the random arrangement of the posts and the relative spatial positioning of the posts throughout the collection region. By pumping the inlet orifice of defined size, a relatively smooth, non-streamlined flow, preferably with an average flow rate of between about 0.1 and 2 mm/s, can be achieved without dead spaces, and more preferably the average flow rate is maintained between 0.2 and 1 mm/s.

In general, the substrate 11 may be made of any suitable material that is acceptable to the laboratory, such as silicon, fused silica, glass, and polymers. It is desirable to use optically transparent materials, which are optionally used, particularly when needed for diagnostics. In its simplest embodiment, the substrate carrying the fabricated microchannels is sealed in a plate 27, as shown in FIG. 3, with the flat surface of the plate 27 facing the surface against which the substrate 11 is to be abutted. The plate can be made of the same material or simply a solid material coated on a glass plate; however, as described below. An intermediate flow regulating plate 25 may be included. Suitable impermeable solid plastics that may be used include: polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), polycarbonate, polystyrene, polyethylene terephthalate, and other well-known polymeric resins acceptable for laboratory use. Substrates of such patterns may be made by conventional methods, such as a method selected from conventional die casting and casting techniques.

Conventional substrates for master or negative mold structures can be made from polymeric materials, and the negative mold structures can be created from thick negative photoresist materials by photolithography, as is well known in the art, and described in the jnanobio technology article, which is incorporated herein by reference. For example, the structural layer may be formed from a commercially available mixture of a standard grade epoxy resin (EPONSU-8) photoresist and hardener (SU-82025) and a photoresist film (e.g., 40 or 50 microns thick) may be spun on a silicon dioxide wafer substrate at 2000 rpm. This thickness determines the height of the flow path in the collection region. The film was pre-baked on a hot plate at a precise level for 3 minutes at 60 ℃ and then for 7 minutes at 95 ℃ to ensure uniform overall thickness, and the resulting sample was cooled at room temperature. The film was exposed to the desired flow path pattern in the final apparatus using a Karl Suss Contact Mask Aligner. The film was then baked at 60 ℃ for 2 minutes, at 95 ℃ for 5 minutes, and then exposed to light in a commercially available SU-8 exposure chamber for 5 minutes with light agitation. This produces a negative mold of photoresist epoxy resin pattern which is used to make a master mold to replicate the pattern post substrate producing PDMA or other suitable polymer resin.

As an example, a PDMA composition was prepared using a 10: 1 weight ratio of PDMA prepolymer to curing agent mixture (Sylgard 184 kit, Corning). The composition is evacuated to remove air bubbles that may form during mixing and then poured onto an epoxy resin master mold placed in a groove of desired depth to form a substrate of desired thickness. The master mold is optionally coated with a thin layer (about 50nm) of a suitable metal (e.g., gold) to facilitate removal of the PDMS replica after curing. The PDMS substrate may be cured at 80 ℃ for 90 minutes, however, the PDMS is first undercured, which may facilitate subsequent functionalization of the collection region (including the pillar surface).

The layout and dimensions of the microchannels 13 and pattern posts depend on the mask used in the exposure step in making the master mold. The depth of the micro-channel 13 is controlled by the thickness of the SU-8 layer of the master mold, depending on the spin-coating conditions. FIG. 2 provides a top view of the microchannel 13 showing the enlarged posts 23 in the collection region 17 arranged in a generally preferred random pattern.

In another embodiment, holes 24 may be drilled or created in the plate without breaking the surface of the removed PDMS template substrate, or drilled in the cover plate to provide inlet and outlet connections. The former, which may be paired with a microscope cover slip or other suitable plate (e.g., a thin sheet of PDMS), provides a non-porous cover or base for the substrate. The two components were subjected to a plasma (resonance) clean for 2 minutes, the cleaned two surfaces were immediately placed in surface contact without touching their facing surfaces, the two facing surfaces were sealed by surface reaction methods well known in the art to form a permanent seal, and the flow path of the microfluidic device was closed.

It is contemplated that SU-8 split master molds incorporating cavities with adjustable flow properties (e.g., pneumatic valves) can be similarly fabricated by processing the fluid streams on an integrated chip of such devices. The flow regulating plate or layer 25 produced from this master mold is first laminated to the microchannel substrate 11 (see fig. 4 and 5) and then to the flat sealing plate 27. Such fluid regulating components and other MEMS employed in microfluidic devices are described in U.S. Pat. Nos. 6,074,827 and 6,454,924, which are incorporated herein by reference. The flow regulating plate 25 was carefully aligned with the substrate 11 carrying the microchannel, and then annealed at 80 ℃ overnight to produce a composite structure. The wells in the flow-regulating plate 25 are then closed with a plate or slide using the same techniques previously described. As a further alternative, a second flow-regulating plate may be laminated to the first plate 25 using the same technique. It is contemplated that more sophisticated controls and optional treatments may be added in this way.

For example, channels may be disposed in the encapsulated substrate fluid regulating layer 25 to form a multi-channel system, thereby providing a chip fluid regulating mechanism. Fig. 4 and 5 illustrate a simple system in which passageways 24a and 24b are connected to the inlet and outlet. Air supplied by pneumatic valve 29 may enter plate 25 through a hole drilled or appropriately formed in substrate 11 or hole 30. The flow-regulating plate 25 and substrate 11 may optionally contain other supply passages for delivering liquid to the inlet 15, and may also contain another outlet or discharge passage as is well known in the art.

As mentioned above, the provision of two serially connected collection regions results in an arrangement which lends itself to different methods of operation and applications. For example, when a sample is to be processed that may contain two different subpopulations of target biomolecules or cells of interest (samples), one type of sequestering reagent may be bound to a pillar in one collection region or chamber and another type of sequestering reagent may be bound to a pillar in a downstream collection chamber. Alternatively, when the target cells are extremely rare, it may be desirable to bind the same sequestering agent to both collection chambers to enhance capture of cells in the liquid sample by perhaps 100%.

From a structural point of view, certain other components that may optionally be incorporated into the device are illustrated in fig. 6 and 7. FIG. 6 shows the incorporation of a set of peristaltic pumps in the flow path connecting the collection chamber inlet and outlet paths in a microchannel similar to that of FIG. 1. The microchannel 13 ' is illustrated as including an inlet 15 ', a collection chamber 17 ' and an outlet 19 ', the integrated pump stack 41 in the chamber containing three specially designed membrane valves added in the inlet path 18 ' to the collection chamber. This figure provides an arrangement similar to that of figures 4 and 5 in which air or other high pressure gas is applied to the passageway 30' causing the sides of the respective valve membranes of the fluid regulating layer or plate to be pressurised, causing the membranes to expand and squeeze liquid in the adjacent region associated with the microchannel. By programming the control unit to operate the three valves in sequence from left to right, the resulting fluctuations pump the liquid in the inlet zone 18 'to the right into the collection device 17'. A set of peristaltic pumps of a similar type may also be incorporated in the outlet zone 45 downstream of the collection chamber 17', if desired.

As another possible alternative, fig. 7 illustrates a micromixing arrangement. The illustrated micromixer 51 includes an annular passage 53 leading to a supply channel 55, which channel 55 may be an inlet channel to the substrate collection chamber described above. A pair of inlet passages 57a and 57b provide liquid to the annular passage 53, and flow of liquid through the passages 55, 57a and 57b is controlled by pneumatic valves 59. Three additional pneumatic valves 61 are located in the channel itself, constituting a peristaltic pump 63 of the type described above. This arrangement provides an efficient method of micro-mixing the two liquids in the substrate itself, which is then transported to a collection chamber or the like. For example, to fill the annular passage 53 with some of the liquid in one inlet channel 57a and some of the buffer liquid in inlet channel 57b, the liquids may be thoroughly mixed by pumping the liquids into the annular passage to flow along the annular passage by sequentially operating the three valves 61 for mixing, and then releasing the mixed liquid through the delivery channel 55.

The polymer surfaces of the patterned post regions can be derivatized in various ways to allow specific sequestering agents for desired target cells or other biomolecules to bind to all of the post surfaces. For example, after the substrate carrying the microchannels has been sealed by plasma (resonance) treatment, the microchannels may be filled with 1-50% volume of an amino-functional silane (e.g., 10% Dow Corning Z-6020 solution), or a thio-functional silane in ethanol, filling the region 17 between the openings 15 and 19, and then placing the filled microchannels 13 in a room temperature incubator for 30 minutes. A polymer that is not fully cured (e.g., PDMS) can be derivatized and then the microchannel regions in the plate sealed. At this point, as noted above, an alternative approach is to first slightly under-cure the PDMS substrate, and then fully cure it after printing the plate and treating it with a substituted silane or other functionalizing agent. For example, the final step of heating at about 50-90 ℃ for about 90 minutes after treatment with Z-6020 may be employed to complete the cure. Alternatively, the curing may be completed by leaving the mixture at room temperature for 1 or 2 days. Derivatization may also be performed prior to sealing the microchannel, since derivatization of the planar surface is not a truly desirable result. After the flow path is flushed with ethanol, the microchannel is ready to receive a sequestering agent that binds the biomolecule.

The term sequestering agent is used to refer to a substance that interacts in a specific manner with a target biomolecule to physically sequester the target molecule. These sequestering agents may include: nucleic acids such as DNA, RNA and PNA bound to proteins, non-hybridizing chelators commonly employed include biological materials such as proteins, e.g., receptors, peptides, enzymes, enzyme inhibitors, enzyme substrates, immunoglobulins (particularly antibodies), antigens, lectins, modified proteins, modified peptides, double stranded DNA, biogenic amines and complex carbohydrates. Synthetic molecules, such as drugs, and synthetic ligands designed to have such specific binding activity may also be used. A "modified" protein or polypeptide refers to a protein or peptide molecule in which one or more amino acids have been changed by the addition of new chemical groups or the removal of certain chemical groups. Or some combination of both such removal and addition. Such changes may include natural and synthetic modifications. Natural modifications may include, but are not limited to: phosphorylation, sulfation, glycosylation, nucleotide addition and lipidation. Synthetic modifications may include, but are not limited to: chemical linkers are added to facilitate bonding with hydrogel, microstructure, nanostructure (e.g., quantum dot) materials, or other synthetic materials. In addition, modifications may include the removal of existing functional groups, such as hydroxyl, sulfhydryl, or phenyl groups, or the removal or alteration of native side chains or the amide backbone of the polypeptide. Examples of complex carbohydrates include, but are not limited to: natural or synthetic linear or branched oligosaccharides, modified polysaccharides (e.g. glycolipids), peptidoglycans, glycosaminoglycans or acetylated sugars, and heterogeneous oligosaccharides such as N-acetylglucosamine or sulfated sugars. Examples of naturally occurring complex carbohydrates are: chitin, hyaluronic acid, keratin sulfate, chondroitin sulfate, heparin, cellulose, and sugar moieties found on modified proteins (such as albumin and IgG). Two or more of such agents may be immobilized on the column in combination, may be added in combination as a mixture of the two components, or may be added sequentially.

The sequestering agent may be directly or indirectly affixed to the posts. The posts may be pre-treated and/or coated to facilitate bonding. Indirect immobilization is obviously preferred, considering the use of an intermediary reagent or substrate first attached to the column; however, it may be desirable to employ a pair of coupling agents to link the chelating agent to the mediating agent. For example, streptavidin or an antibody directed against another antibody (Ab) can be linked to an intermediary reagent, which in turn is coupled to a biotinylated Ab or an antibody directed against another Ab. This arrangement allows the gene products of the microfluidic device to be used to capture a variety of cells in different samples, or to perform subtractive enrichment.

Cell separation and adhesion preferably utilizes abs as the chelating agent, as described in U.S. patent nos. 5,646,404 and 4,675,286 and prior art. For example, U.S. Pat. No.4,528,267 describes a non-covalent binding method. Ichirochiba in IMMOBILIZED ENZYMES; halstead Press: new York (1978) and in a. cuatrecasas, J Bio chem.245: 3059, 1970, which is incorporated herein by reference. Kawata et al, J Exp Med.160: 653, 1984, describe methods for isolating placental cell populations by detecting target cells using cell-specific abs, such as anti-human trophoblast monoclonal antibodies (anti-Trop-1 and anti-Trop-2). U.S. Pat. No.5,503,981 identifies three other monoclonal antibodies that can be used for this purpose.

The antibody is preferably bound to the solid phase surface of the posts by an indirect method, such as by using a surface layer or coating layer containing a long linker to which the Ab can bind. For example, the surface may be coated with a bifunctional or multifunctional reagent (e.g., a protein) and then the reagent may be conjugated to the antibody using a coupling agent (e.g., glutaraldehyde). The antibody can also be effectively bound by applying an aqueous solution of the antibody to a surface that has been coated with a layer of free isothiocyanate or equivalent group (e.g., isothiocyanate polyester), or the antibody can be coupled to the hydroxylated material using cyanogen bromide. It is particularly preferred to use a hydrophilic polyurethane hydrogel layer (containing free isothiocyanate groups) as described below in connection with fig. 9, or to use a hydrophilic linker of suitable length, such as PEG, polyglycine, as described below in connection with fig. 10.

The chelating agent is chosen to specifically capture the biomolecule of interest, and the target molecule can be various cells, as well as proteins, viruses, sugars, etc.; however, it is believed that the present invention shows particular efficacy and particular advantages in isolating cells. Although the term "cell" is used in this application, it is to be understood that it includes fragments and/or fragments of the cell carrying the chelator-specific surface ligand. As is known in the art, the desired specificity for a target biomolecule can be obtained by selecting suitable chelating agents with specific high affinity. As described above, the microfluidic device can also be used for subtractive enrichment by targeting known contaminating cells.

When an antibody is used, it may be suitably bound by a mechanism well known in the art, preferably through an intermediary reagent. For example, Ab was thiolated by treatment with 2-aminosulfane, and the resulting thiolated antibody was coupled to a post that had been treated with PEG-maleimide; alternatively, the antibody may be directly bound to a suitable reactive hydrophilic isothiocyanate group or thiocyanate group.

After the antibodies or other chelating reagents are disposed throughout the post collection regions of the pattern, the microchannel device is ready for use. Bodily fluids (e.g., blood, urine samples) or some other pre-treated liquid containing the target cell population are carefully discharged from a standard syringe pump into the inlet channel 24a leading to the inlet 15 of the microchannel device, or pumped by a vacuum pump or the like, to flow along the flow through the collection region 17, the larger diameter inlet channel 24a providing a sample storage well for retaining the volume of sample required for testing. In use, the opening 24a may contain a fitting (not shown) that mates with tubing connected to the syringe pump. The pump is operable to produce a flow of about 0.5 to 10 microliters/minute through the device. Depending on the bodily fluid or other cell-containing fluid to be treated and/or analyzed, a pretreatment step may be employed to reduce its volume and/or delete unwanted biomolecules, as is known in the art.

In order to greatly increase the overall efficiency of the cell separation method, it is necessary to collect the sample flowing out of the outlet 19 and to (repeatedly) flow it through the microchannel more than once; such repeated treatments may be particularly useful when the cells are particularly rare and in very small numbers in the sample. However, such repeated flows are expected to be occasionally required due to the high capture efficiency achieved by the device. Alternatively, two collection chambers can be used in series as described above. However, if the body fluid sample to be processed is of a large volume, two or more parallel microchannels in the substrate may be used.

The chelating agent can be bound to the bottom, facing surface, pillars and sidewalls of the microchannel collection chamber. However, the side walls do not disrupt the flow as effectively as the floor, facing surface and pillars. It has been determined that when a liquid containing cells or other biomolecules flows uniformly through the collection chamber, the cells reside primarily in the central flow region where shear forces are minimized; thus, the capture of the sidewall with the sequestering agent is considerably less than the capture of the surface of the immediate area across which the posts are positioned, thereby disrupting the streamlined flow. In these regions, the chelating agent ensures its natural three-dimensional conformation, due to the correct coupling, and the effect is surprising.

After the liquid sample has been allowed to flow completely through the device, the target cells, if present, are captured in the collection region, and the collection region is first washed with a buffer to remove any extraneous biological material that is part of the sample but that is not strongly captured by the antibody or other chelator. It is expected that only target cells bound in the collection region of the microchannel device will remain after washing with an effective buffer, while any non-specifically bound material is removed.

Once the buffer wash is complete, the cells can then be released appropriately if the purpose of the treatment method is simply to collect the cells. As described below, in some cases, it may be desirable to perform some analysis in situ. For example, bound cells in the collection chamber or downstream region may be counted, or the cells may be lysed for PCR.

When cells are to be released, methods known in the art may be employed, such as mechanical methods (e.g., high velocity liquid flow), chemical methods (e.g., changing pH), or the use of enzymatic cleavage agents, among others. For example, a reagent may be added to cleave the chelator or cleave the linkage between the chelator and the cell to release the target cell from the collection region. For example, trypsin or specific focusing enzymes can be used to degrade abs and/or cell surface antigens. Specific methods for binding abs and the like and then effectively removing the captured ligand are described in U.S. Pat. No.5,378,624. For example, if the cells have been bound by the rare cell surface specific antibodies used, they can be released by treatment with trypsin or other suitable proteases (e.g., proteinase K). Alternatively, collagenase may be used to release the other chelator, or a specifically cleavable linker may be used to bind the chelator. During cutting, the microchannel outlet is connected to a reservoir or other collector, and the effluent containing released cells is collected for further analysis. The manufactured micro-channel can be provided with more than one discharge passage and outlet and a valve for adjusting the opening of the outlet; thus, one outlet path can be used to release waste during the preliminary experimental steps and another outlet path can be used to allow the target cell fluid to flow into the collection container.

It has been found that the arrangement and shape of the posts 23 in the patterned post collection region 17 can be engineered to optimize fluid dynamics and enhance capture of target cells by virtue of their surface characteristics. Very generally, in most cases, the horizontal cross-section of the transverse fixed posts 23 is preferably shaped to avoid sharp corners, which may promote non-specific binding of the lateral surfaces of the posts. The posts 23 may have rectilinear outer surfaces, preferably of generally circular cross-section or of regular polygonal shape with 6 or more sides. Alternatively, a useful shape is a teardrop shape, with the tip slightly curved downstream, or an oval shape; however, if more impact is desired, a square shape may be used. The pattern of pillars should produce a flow pattern in the fluid stream that enhances capture of target cells by the sequestering agents bound to the pillar surface, bottom surface and facing surface. To accomplish this, it has been found that the posts should be of different sizes and arranged in a random pattern. Surprisingly, random patterns of posts 23 of varying cross-sectional sizes, e.g., at least 3 or 4 different sizes, with a diameter of about 70-130 microns, and a collection region of about 100 microns in height and about 2-4 microns in width, appear to be particularly effective in capturing cells in a flowing liquid sample, with a minimum spacing between the posts of 50-70 microns, preferably about 60 microns.

It is particularly preferred that the cross-sectional area of the post region, i.e., the area bounded by the side walls formed by parallel lines perpendicular to the floor, occupy about 15-25% of the volume of the collection region. Preferably, the pattern of posts occupies about 20% of the volume of the collection region, leaving about 80% of the volume of the void for fluid flow. The specific random pattern of post locations shown in FIG. 2 appears to particularly enhance the propensity of cells to be captured by the sequestering agent since streamlined flow in these regions has been effectively disrupted.

Smaller posts downstream of larger posts may create vortex regions and thus flow patterns, indicating that the capture of target cells at the surface of adjacent regions, particularly at the bottom of the collection region, is particularly effective. As shown in fig. 2, the flow path extends longitudinally in a straight line more than about 100 microns from the sidewall across the plurality of posts. As noted above, the posts are integral with the substrate bottom surface 20, preferably with their opposite or free ends fixed to the facing surface, i.e., to the flow-regulating plate 25 or to the sealing plate 27.

As described above, a chelating agent (e.g., an antibody) can be incorporated throughout the collection region by coating the surface with a layer of a specialized hydrophilic hydrogel material or (b) a hydrophilic linker, such as PEG, polyglycine, or the like, having a molecular weight of at least about 1000 daltons, preferably a molecular weight of between about 2000-. It is particularly preferred to coat with (a) a permeable hydrophilic hydrogel which is a polymer of PEG, PPG or a urethane-bonded copolymer thereof containing isothiocyanate functional groups and containing reactive isothiocyanate groups. Preferred hydrogels employ a three-branched PEG with a molecular weight of about 6000, which can be produced by adding ethylene oxide to glycerol. The resulting polyol was reacted with isophorone diisothiocyanate and trimethylolpropane to prepare a prepolymer. The prepolymer is crosslinked in situ on the surface of the collection region of the microfluidic device in a mixture with appropriate buffers, solvents and other components for the particular application. The schematic shown in FIG. 8 provides a microchannel collection region having a plurality of posts 61 of different diameters arranged randomly to disrupt streamlined flow through the chamber, each post 61 and the facing planar surface having an outer coating 63. A chelating agent 65 in the form of an antibody is bound to the permeable hydrophilic hydrogel coating on the posts; the chelating agent thus retains its native three-dimensional conformation and is not altered by binding to the hydrogel, whose main component is water, with little deformation.

Fig. 9 provides a schematic illustration of the chemicals that may be used when employing the performance preference permeable hydrophilic hydrogel coating 49 of fig. 8. Representative sequences of chelating agents (i.e., antibodies) that bind to all surfaces of the collection region are shown. Point 1 in fig. 9 represents the surface after amino derivatization with aminosilane or the like. This step is followed by a surface coated with skimmed milk (blocking) casein, see point 2. Point 3 represents the coated surface after coating. PEG prepolymers containing a molecular weight of about 3400 are dissolved in a water-miscible organic solvent (preferably an aprotic solvent such as NMP and CH)₃CN mixture) was blocked with toluene diisothiocyanate. Preferably the polymer contains a functional triol or higher alcohol, such as PEG and PPG, which may contain a trifunctional isothiocyanate. An aqueous solution containing 98.5% water is then prepared and pumped through the microchannel, and the surfaces of the posts and facing surfaces of the collection region are coated with this hydrophilic hydrogel coating as a result of reaction with certain end-capped isothiocyanate groups on the amino-derivatized surface. The final result is shown in FIG. 9, point 3, where the hydrogel produced was formed by first reacting with water and then forming urea bonds.

Point 4 shows the addition of an antibody containing surface amino groups. As shown in point 5, this antibody can be directly bound to the permeable hydrophilic hydrogel coating of the pillars by covalently binding the amino group of Ab to the isothiocyanate or thiocyanate group contained in the hydrophilic coating. Alternatively, the antibodies can be thiolated first as shown at point 6 in FIG. 9, and then the aqueous thiolated antibodies can be supplied to the collection chamber, which in turn facilitates covalent bonding to the isothiocyanate groups of the coating polymer, see FIG. 7.

As shown in fig. 8 and 9, when the cells in the liquid sample are caused to flow through the collection chamber, the cells contact the posts and/or face the surface, the specific antigen of the antibody on the cell surface binds thereto, and the cells are effectively captured, due to disruption of the streamlined flow.

FIG. 10 provides a schematic representation of representative chemical reagents that may be employed when an elongated PEG or PPG linear polymer is used in place of a hydrogel to attach a chelator, particularly an antibody, to the surface of the collection area. The linear polymer length is chosen such that the antibody retains its native three-dimensional conformation in the aqueous environment at the time of capture. Point 1 of fig. 10 shows the surface derivatized with amino groups by treatment with aminosilane or the like. This step was also followed by (blocking) the casein-coated surface with skim milk as described above. After washing all surfaces are treated with linear PEG or PPG (which has a NHS group at one end and a maleimide group at the other) with a molecular weight of at least about 2000, preferably about 3000. The N-hydroxysuccinimide ester group readily reacts with amino groups on the surface to produce a coating at least about 1 micron thick, and after appropriate incubation the microchannel is flushed with an appropriate buffer, leaving a maleimide-PEG coated surface at point 3 of FIG. 10. Point 4 shows the trophoblast cell-specific antibody and the intrinsic surface amino groups, and the antibody is thiolated with a suitable reagent (e.g., Traut reagent) to react with the spots shown in FIG. 10, point 5. The purified thiol antibody buffer solution is introduced into the microchannel for appropriate incubation to couple the thiol antibody to the maleimide-PEG-coated posts. The microchannel is washed with an appropriate buffer and the coupling arrangement obtained is at point 6.

FIG. 10 provides a schematic at point 7 showing the capture of trophoblast cells by antibodies attached to the surface via a linear PEG coupling agent.

Another more preferred configuration of the microfluidic device is shown in fig. 12-15. The microfluidic device shown is similar to that shown in figures 1-3 but has the structural advantage that the uniform flow in the collection region, i.e. by gravity, improves contact between the liquid to be treated and the internal surfaces of the device, thereby minimising adhesion of target cells outside the collection chamber region (e.g. the inlet and outlet). In this regard, the device is designed with a tendency to use an angle of about 30-60 degrees, preferably 45 degrees, from horizontal. Thus, the device comprises a body or substrate 73 containing microchannels 75 extending from an inlet passage 77 to an outlet passage 79, and a collection zone 81 located between the passages, the collection zone 81 comprising an inlet zone 83 and an outlet zone 85 as described above. The collection region 81 forms a recess or receiving chamber in the planar surface 87 of the body. The collection region itself has a planar bottom surface 89 that is substantially parallel to the body surface 87. The plurality of posts extend as described above along a bottom surface 89, perpendicular to the bottom surface, parallel to the main body plane 87, and together separate the streams of liquid that are routed into and out of the zone.

The structure of the device 71 is substantially the same as that described above with respect to fig. 1-5, except that the inlet and outlet passageways 83, 85 are outwardly directed. As shown in fig. 12 and 14, the cavities that make up the microchannels 75 are sealed with a solid plate 93 that is preferably larger in size than the body to facilitate the operation of the overall device during liquid sample processing and analysis. The closure plate 93 may be made of glass or a suitable impermeable polymer material as described above. The plate 93 may be coated with a layer of the same polymer used to cast, mold the body, or be appropriately constructed. More preferably, a PDMS polymer is used, and if a standard 25mm by 75mm glass slide is used, the slide may be coated with a layer of PDMS or film 95 as shown in FIG. 12. The plane of the closing plate 93 is glued to the end surface of the above-mentioned upright 91, or simply made substantially contiguous.

As best shown in fig. 14, the inlet passages 77 are arranged at an acute angle of 30-60 degrees, more preferably 40-50 degrees, and most preferably 45 degrees from the planar surface of the microchannel parallel to the entire flow path. This arrangement allows the feed sample to flow vertically down into the microfluidic device 71 as shown in figure 15, with the plate 98 confining the (liquid in) the device to an inclined surface along its upper edge towards the base 96, with the overall flow path inclined (e.g. at a 45 degree angle) to be vertical. The feed sample thus fills the wide entry region 83 to the collection region 81, gravity helps it remain in the collection region containing the posts, filling and promoting the desired slow uniform flow in this region. More importantly, because the liquid flows linearly through the chamber in a direction parallel to the upper and lower surfaces, and because the cells are heavier than the aqueous buffer solution in which they are carried, the force vector generated by gravity on these cells is different from the force vector generated on the carrier liquid. Thus, in addition to the disruptive effects of the different size random pattern of posts in the collection region on the streamlined flow stream, the force vectors can cause cells to leave the stream and adhere to the surface by binding to the sequestering agent. This effect was found to result in a slower flow rate through the collection region, which greatly improves cell collection.

The inlet and outlet passageways 77, 79 are preferably arranged in the same vertical plane, with the outlet passageways preferably oriented at about 90 degrees. This allows the device to be clamped to the base 96 as shown in figure 15 with the inlet passageway 77 vertical and the outlet passageway 79 horizontal. This allows the cells and other biological materials contained in the sample to flow horizontally without being bound by the Ab, facilitates their removal from the fluid channel and minimizes non-specific adhesion, and settles in the outlet zone.

Figure 15 also shows a preferred method of processing samples added vertically to the access to the microfluidic device inlet. The outlet passage 79 is connected to the pump suction side of the syringe by suitable tubing to facilitate the flow of sample through the device at the desired flow rate. The flow rate at which the fluid sample is discharged at a rate equal to the average rate of flow of fluid into the collection region, between about 0.1 mm/sec and about 1 mm/sec, preferably between about 0.2 mm/sec and about 0.8 mm/sec, and more preferably between about 0.25 mm/sec and about 0.5 mm/sec, is carefully controlled to maximize the capture of target cells due to the force vector created by the column's disruption of fluid flow and gravity. High flow rates have been found to be less effective and may also damage delicate cells sought. While the device is shown in its simplest form, various valves and ancillary components known in these MEMS devices and described herein above may be assembled with the microfluidic (bed) device 71.

The following example illustrates that prototype microchannel devices of this type can be effectively used to sequester (capture) trophoblast cells in cervical mucus extracts. The flow chart attached to fig. 11 summarizes the overall method. These examples should, of course, be understood as merely illustrative of certain embodiments of the invention and not as limiting the scope of the invention, which is defined by the claims at the end of this specification.

Example 1

A microfluidic device for separating biomolecules was constructed using a prototype substrate generally as shown in fig. 1. A substrate formed of PDMS was bonded to a glass plate to seal the fluid channel. The inner surfaces of the entire collection region were derivatized by incubation with 10% by volume Dow Corning Z-6020 solution at room temperature for 30 minutes. After washing with ethanol, treatment with skim milk for about 1 hour at room temperature yielded a casein thin coating. After washing with 10% aqueous ethanol, the isothiocyanate-terminated PEG triol prepolymer having an average molecular weight of 6000 was treated by mixing one part by weight of this prepolymer and 6 parts by weight of organic solvents (i.e., acetonitrile and DMF) with water to form a permeable hydrogel, and coating the inner surface thereof by passing it through the channels as shown in FIG. 9.

To perform this assay, trophoblast cells are isolated from a cervical mucus sample and anti-Trop-1 and Trop-2 antibodies specific for ligands carried on the outer surface of trophoblast cells from embryos are selected. The antibody was dissolved in 100. mu.l of 0.2M sodium borate/0.15M NaCl containing 5mM EDTA (pH8.3) and reacted with 5. mu.l of 40mM Traut reagent at room temperature for 1 hour to carry out thiolation. Excess Traut reagent was reacted with 10. mu.l of 100mM glycine, followed by reaction at Centricon-30^TMThiolated antibody was purified on a membrane. Thiolation was verified using standard laboratory methods.

About 5 micrograms total of thiolated Trop-1 and-2 antibody aqueous solution at a concentration of about 0.5mg/ml is added to the pretreated fluidic device, the solution is incubated at 25 ℃ for 2 hours, and after incubation, the fluid channel-producing antibody-coated surfaces are rinsed with 1% PBS/BSA for isolation of embryonic trophoblast cells.

Cervical mucus from pregnant mothers (8-12 weeks gestation) was diluted to 10ml with HAM solution (Vitrogen), treated with dnase at 37 ℃ for 30 minutes, filtered through a 100 μm cell filter, and then centrifuged at 1500rpm for 30 minutes. The pelleted cells were resuspended in HAM solution 100. mu.l and the cell suspension was flowed through Trop-1 and Trop-2 antibody-coated microchannels by connecting the microfluidic separation device to the delivery line of a Harvard device syringe pump (containing about 50. mu.l of cell suspension of cervical mucus extract). The syringe pump was pushed at room temperature to slowly and continuously flow the liquid sample through the microfluidic device at a flow rate of about 10 microliters/minute. At this point, the Trop-1 and Trop-2 antibodies, which have bound to the randomly distributed pattern of transverse posts in the collection region, capture trophoblast cells present in the sample. After pumping all samples with a syringe pump, the samples were rinsed slowly with 1% PBS/BSA buffer. About 100. mu.l of this buffer was fed into the device for about 10 minutes, effectively removing all non-specifically bound biological material in the fluid channels of the device. Then, the washing is performed twice, each time with about 100. mu.l of 1% PBS and 1% BSA for about 10 minutes.

At this time, the capturing effect was examined by an optical microscope as in the apparatus made of an optically transparent material. Bound cells were stained with cytokeratin 7 and cytokeratin 17 (antibodies) characteristic of the captured trophoblast-derived cells. The cells were counted by light microscopy and the cells captured by the patterned post collection region in the sample were estimated to be substantially 97% trophoblasts. This result was considered to be excellent.

The entire capture and wash procedure was repeated without in situ staining, and 100. mu.l of a 0.25% trypsin solution was slowly flowed through the fluidic channel at 27 ℃ for 20 minutes to release the captured trophoblast cells. The agent digests the Ab and releases trophoblast cells into the water stream, which is collected as the water stream flows to the outlet. Analysis of the collected cells using PCR and FISH based techniques showed that it was indeed the trophoblast cells targeted by the antibody used.

Example 2

Another microfluidic device for separation of biomolecules was constructed using a prototype substrate as described in example 1. The inner surface of the substrate was derivatized as described in example 1, washed with ethanol, and treated with skim milk. After washing with 10% aqueous ethanol, the prepolymer was treated with BSA and Trop-1 and Trop-2 antibody borate buffer. A1 mg/ml aqueous antibody solution was prepared using sodium borate pH8.0100mM containing BSA. The special preparation contains 100mg of the same prepolymer dissolved in Acn/DMF; 350. mu.l of boric acid buffer containing 0.25mg/ml of antibody; 350. mu.l of 1mg/ml BSA borate buffer containing about 20% by weight of polymer.

Antibodies were not thiolated and a total of 5. mu.l of Trop-1 and Trop-2 hydrogel solutions were added to the pretreated microfluidic device. The solution was incubated at 25 ℃ for about 30 minutes, after which the irrigation fluid channel was slowly pushed into the fluid channel with mineral oil to displace the hydrogel and push out the excess hydrogel. The result of the filling of the fluid channels with oil is the separation of the thin hydrogel coating from the PDMS material. After 3 hours the hydrogel was fully cured and the oil was drained by rinsing with 1 xPBS/0.1% Tween solution. The device was then filled with 1xPBS solution to preserve the Ab.

The cervical mucus from the pregnant mother was diluted, treated, filtered, centrifuged, and resuspended in 100. mu.l HAM solution. The cell suspension samples were passed through the Trop-1 and Trop-2 coated microchannels using a syringe pump in a Harvard apparatus as in example 1. After pumping all sample solutions with a syringe pump, the samples were slowly rinsed with 1% PBS and 1% BSA buffer. Approximately 100 μ l of the buffer was allowed to flow through the device for approximately 10 minutes, effectively removing all non-specifically bound biological material in the fluid channels of the device. Then, the washing is performed twice, each time with about 100. mu.l of 1% PBS and 1% BSA for about 10 minutes.

After staining the bound cells with cytokeratin 7 and cytokeratin 17 (antibodies), the effect of capture was examined by light microscopy. The cells were counted using an optical microscope and it was determined that excellent capture of trophoblast cells present in the sample was achieved.

Example 3

Another microfluidic device for separation of biomolecules was constructed using a prototype substrate as described in example 1. The inner surface of the substrate was derivatized as described in example 1, washed with ethanol, and treated with skim milk.

After washing with 10% aqueous ethanol, 10. mu.l of a 2.5mM NHS-polyglycine (average molecular weight about 4500) solution prepared with 0.2MOPS/0.5M NaCl, pH7.0 was pumped through the channel gently back and forth for 2 hours. The micro-channel was washed three times with 500. mu.l of MOPS buffer at pH7.0 to obtain a maleimide-polyglycine-coated channel.

Trophoblast cell outer surface ligand-specific antibodies Trop-1 and Trop-2 were treated to thiolate as described in example 1.

An aqueous solution of thiolated Trop-1 and-2 antibodies at a concentration of about 5 micrograms total of about 0.25mg/ml is added to the pretreated microfluidic channel, the solution is incubated at 25 ℃ for 2 hours, and after incubation, the fluid channel is rinsed (three times) with 1% PBS/BSA to produce antibody-coated surfaces, which are then used to isolate embryonic trophoblasts.

The cervical mucus from the pregnant mother was diluted, treated, filtered, centrifuged, and resuspended in 100. mu.l HAM solution. The cell suspension samples were passed through the Trop-1 and Trop-2 coated microchannels using a syringe pump in a Harvard apparatus. After all samples were pumped with a syringe pump, they were slowly rinsed with 1% PBS and 1% BSA buffer. Approximately 100 μ l of the buffer was allowed to flow through the device for approximately 10 minutes, effectively removing all non-specifically bound biological material in the fluid channels of the device. Then, the washing is performed twice, each time with about 100. mu.l of 1% PBS and 1% BSA for about 10 minutes.

After staining the bound cells with cytokeratin 7 and cytokeratin 17 (antibodies), the effect of capture was examined by light microscopy. The cells were counted using an optical microscope and it was determined that excellent capture of trophoblast cells present in the sample was achieved.

Example 4

A plurality of microchannels similar to those used in example 1, as shown in FIG. 3, were prepared to examine whether the results improved when operated at a 45 degree angle to the horizontal. The added liquid used a mixture of BeWo and Jurkat cells and examined the effect of microfluidic device 71 positioned at this angle for improvement. BeWo cells were chosen because they expressed the Trop-1 and Trop-2 antigens, while Jurkat cells, which did not express either antigen, served as negative controls. The inner surface of the microfluidic channel 71 was pretreated and then coated with permeable hydrogel as described in example 2, and the coating solution used was an aqueous solution of anti-Trop-1 and Trop-2 (antibodies). The fluid channel inner surface was filled with the Ab coating liquid and incubated at 25 ℃ for about 30 minutes. As in example 2, the cells were washed with mineral oil and then PBS buffer.

A sufficient amount of test solution for 6 tests was prepared. It contained about 3000 BeWo cells and about 3000 Jurkat cells in 1% BSA/PBS buffer. The solution was divided into 6 aliquots, each containing about 500 BeWo cells and about 500 Jurkat cells. Three identical microfluidic devices 71 were horizontally positioned and each aliquot of mixed cell fluid was pumped through each device using a vacuum pump. Three test units used three different flow rates: 1 microliter/minute, 5 microliter/minute and 10 microliter/minute.

After flowing through the three test devices, the devices were washed with PBS buffer and examined microscopically. Two groups of captured cells were counted separately under the mirror. For the BeWo target cells, it was found that at the lowest flow rate approximately 47% of the BeWo cells were captured in the entrance zone, only approximately 32% of the collection channels were captured, and the remaining cells were in the exit zone. At a flow rate of 5 μ l/min, the percentage of BeWo cells captured in the collection region decreased slightly to about 27%, while more cells were still captured in the inlet region, with the highest percentage being collected in the outlet region. The collection region captured only 10% of the BeWo cells when the device was traversed horizontally at the highest flow rate, while the exit region collected approximately 65% of the cells. For Jurkat cells, the inlet and channel regions each captured about 20-25% of the cells at the lowest flow rate, and the collection channel region captured only about 10% of the Jurkat cells at the intermediate flow rate. As expected, the number of cells collected in the outlet zone increased with each increase in flow rate, mixing all cells not retained by the collection device.

The experiment was repeated with three identical fluidic devices each at a 45 degree angle to the vertical. Flow rates of 1, 3 and 5. mu.l/min were used at this time. Surprisingly, the capture of desired BeWo cells in the collection channel region is enhanced. At the lowest flow rate, the collection channel region captured about 75% of the BeWo cells. This increased to 82% at a medium flow rate of 3 μ l/min, and about 60% at a maximum flow rate of 5 μ l/min was still tested. On the other hand, although the non-specifically bound Jurkat cells in the collection region were higher at the lowest flow rate, about 45%, the 3. mu.l/min flow rate dropped to about 15% and less than 5% at the highest flow rate. Thus, when operating at a flow rate of 3-5 microliters/minute, the performance improvement is very large at a 45 degree angle to the horizontal, with about 80% of cells collected rather than just 27%. Calculations show that by manipulating the flow rate to equal about 0.27 mm/sec through the collection chamber region, excellent collection of target cells is achieved with minimal contamination of non-specifically bound cells.

While the invention has been described with respect to certain preferred embodiments, which constitute the best modes presently known to the inventors for carrying out the invention, it should be understood that various changes and modifications as would be obvious to one having ordinary skill in this art may be made without departing from the scope of the invention which is defined in the claims which follow. For example, although certain preferred materials have been described for use in fabricating microchannel substrates, many structural materials are known in the art to be useful and are suitable for use as such laboratory devices. Although the present disclosure is generally directed to isolating fetal cells from a maternal blood sample and isolating trophoblast cells from a cervical mucus extract, it is to be understood that the present invention may be used to isolate a wide variety of blood cells, such as anucleated red blood cells, lymphocytes, metastatic cancer cells, stem cells, and the like; other biological substances, such as proteins, sugars, viruses, etc., in the liquid sample may also be isolated. When the sample contains a particular subpopulation of cells, the target cells to be captured may be a group of cells that need not be separated from rare cells. However, once the target cells are collected, they can be lysed in situ to provide cellular DNA for downstream analysis or PCR in the collection chamber. U.S. patent application 2003/0153028 describes lysis of bound cells to obtain their released nucleic acids. If two different subpopulations of target cells are present in the sample, different chelators can be bound to the posts in a pair of upstream and downstream collection chambers. Alternatively, it is preferred to collect a cell in the upstream collection chamber, release it, and screen and isolate a subgenus of cells in the downstream collection chamber.

Claims (1)

1. A microfluidic device for separating biomolecules in a bodily fluid or other liquid sample, said device comprising:

a body having a flow path defined as a cavity in a planar surface through which a sample containing a target biomolecule can flow, said flow path comprising an inlet means, an outlet means and an arrangement of microchannels extending between said inlet and outlet means, and

the sealing plate device is provided with a sealing plate device,

the planar surface of the seal plate means is adjacent the planar surface of the body and proximate the flow path cavity,

wherein said microchannel arrangement comprises a collection region having a plurality of transversely spaced posts integral with the bottom surface of said collection region and extending therefrom to the surface of said sealing plate means, wherein the posts are arranged in a random pattern and extend laterally across said flow path in said collection region, all surfaces of said collection region comprising said posts coated with a permeable hydrophilic hydrogel and carrying a chelating agent to which a target biomolecule is to be bound, said posts having a cross-section which occupies between 15 and 25% of the volume of said collection region.

2. The apparatus of claim 1, wherein said columns are arranged substantially perpendicular to said bottom surface.

3. The apparatus of claim 1, wherein the posts have at least 3 different cross-sectional dimensions.

4. The device of claim 1, wherein the diameter of the posts is 70-130 microns.

5. The apparatus of claim 1, wherein the minimum distance between two of said posts is 50-70 microns.

6. The apparatus of claim 1, wherein the inlet means comprises an inlet passage at an angle of 30-60 degrees to the bottom surface.

7. The apparatus of claim 1, wherein the inlet means and the outlet means comprise an inlet passage and an outlet passage, respectively, and the inlet passage and the outlet passage are arranged at a 90 degree angle to each other.

8. The apparatus of claim 1, wherein the outlet means comprises a horizontal outlet passage.

9. The apparatus of claim 1, wherein the outlet means is connected to a pump.

10. The device of claim 1, wherein the body has a planar surface and an arrangement of microchannels in the flow path chamber below the planar surface.

11. The device of claim 1, wherein the surfaces of the posts and the collection region are coated with a permeable hydrophilic coating.

12. The device of claim 1, wherein the surfaces of said posts and said collection region are coated with a permeable hydrophilic hydrogel coating at least 1 micron thick and formed from an isothiocyanate functional prepolymer produced by reacting PEG, PPG, or a copolymer thereof with a polyisothiocyanate.

13. The device of claim 1, wherein said posts and said collection region surface are coated with a chelating agent.

14. The device of claim 1, wherein the posts and the collection region surface are coated with a chelating agent selected from the group consisting of antibodies, antigens, receptors, peptides, drugs, nucleic acids, lectins, enzymes, enzyme inhibitors, enzyme substrates, modified proteins, modified peptides, biogenic amines, complex carbohydrates, synthetic ligands, and combinations thereof.

15. The device of claim 1, wherein said chelating agent is coupled to the surface of said posts and said collection region via a linker.

16. The device of claim 1, wherein the chelating agent is coupled to the surface of the posts and the collection region by a hydrophilic linker or a hydrophilic gel coating.

17. The device of claim 1, wherein the inlet device comprises a well capable of receiving a liquid sample.

18. The device of claim 1, wherein the microchannel is sealed with a plate attached to the free end of the posts.

19. The device of claim 1 wherein said microchannel is sealed with a plate attached to the free end of said pillar, said plate containing a fluid regulating layer.

20. The device of claim 1, wherein the microchannel comprises an optically transparent bottom surface and is viewable with an optical detection device.

21. The device of claim 1 wherein said microchannel is sealed with a plate attached to the free end of said posts, said bottom surface of said collection region and said plate being optically transparent.

22. A method of capturing a target molecule in a sample, the method comprising flowing a liquid containing the sample through the posts and the collection region of the device of claim 1, wherein the surfaces of the posts and the collection region are coated with a chelating agent capable of binding to the target molecule.

A method of detecting a target molecule in a sample, the method comprising:

flowing a liquid containing said sample through the posts and said collection region of the device of claim 1, the surfaces of said posts and said collection region being coated with a chelating agent capable of binding to the target molecule, and

detecting the target molecule.

24. The method of claim 22 or 23, wherein the target molecule is a cell.

25. The method of claim 22 or 23, wherein the target molecule is a blood cell, a metastatic cancer cell, or a stem cell.

26. The method of claim 22 or 23, wherein the target molecule is a fetal cell.

27. The method of claim 22 or 23, wherein the liquid containing the sample flows through the microchannel at a velocity of 0.2 to 1 mm per second.

28. The method of claim 22 or 23, wherein the liquids are mixed and then flowed through the microchannel.