WO2000063684A1

WO2000063684A1 - Charge-flow separation devices and methods

Info

Publication number: WO2000063684A1
Application number: PCT/US2000/010792
Authority: WO
Inventors: Hyoun Park; Zilan Shen
Original assignee: LEU TZONG-SHYNG (JEREMY); Sarnoff Corp
Current assignee: LEU TZONG-SHYNG (JEREMY); Sarnoff Corp
Priority date: 1999-04-21
Filing date: 2000-04-21
Publication date: 2000-10-26
Anticipated expiration: 2001-10-21
Also published as: WO2000063684A9; WO2000063684A8

Abstract

A separation device (60) comprises a separation channel (61) having a width and a height, liquid first inputs (62) at the first end, liquid first outputs (64) at the second end, sources of traverse forces comprising traversing liquid inputs (65) and liquid outputs (66) adapted to provide liquid flow along an axis intersecting flows from the first inputs (62) to the first outputs (64) and a pair of electrodes (68, 69) providing an electrical field across the width of the channel (61) wherein the device further includes two or more traversing liquid inputs (70) and liquid outputs (71) comprising channels through a substrate in which the separation channel (61) is formed and the channels opening in the channel on a top or a bottom surface of the channel wherein the openings are interior to the electrodes (68, 69) or other various features.

Description

CHARGE-FLOW SEPARATION DEVICES AND METHODS

This application claims benefit to US Provisional Patent Application Number 60/130,325 filed April 21, 1999.

The present invention relates to devices and methods for separating molecular, aggregate or cellular species, typically charged species, in a charge-flow separation devices. Charge-flow separation devices have been used to separate minor sub-populations of cells from a mixed population of cells. For example, such devices can be used as part of a protocol for isolating nucleated fetal blood cells from the mother's blood cells. The method thereby allowing biochemical, including genetic, analysis of fetal tissue while avoiding invasive efforts to obtain such tissue. Charge-flow separation devices can be used to separate other substances, particularly those that sediment to some degree in the separation medium. Such charge-flow separation ("CFS") devices are described, for example, in US Patents 5,948,278, 5,906,724, 5,676,849, 5,662,813, 5,439,571, 5,336,387 and 5,173,164.

CFS devices operate by injecting/inserting sample to a broad channel through which a flow is maintained from an inlet (insertion) end to an outlet end. While the sample moves from the inlet end to the outlet end, a traverse force acts on the analytes. (Note that the term "analyte" is used for convenience, though CFS devices are often used in a preparative mode to isolate as well as analyze substances or cells.) The traverse force can be a flow across an intersecting flow path from that defined by the axis from the inlet end to the outlet end, an electric field, or both. For Example, in CFS device 20, illustrated in Figure 1, liquid separation medium is inserted at inlets 12, for example at flow Fi, and withdrawn at outlets 13, to provide "direct" flow into separation channel 10. The sample is inserted at an inlet selected as appropriate given the character of the separation to be obtained, as determined from test operations. Si and S₂ illustrate sample insertions. Barrier/membrane 11 is designed to allow traverse fluid flow, while reducing convection currents and other mixing forces. As illustrated, the membranes 11 are compression seated between elements of the substrate forming the CFS device 20. One source of traverse force is provided by flow from countercurrent inlets 18 to countercurrent outlets 19. Countercurrent inlets 18 receive liquid from inlet manifold 16, while countercurrent outlets 19 receive liquid from outlet manifold 17. For example, flow F₂ is applied to the inlet manifold 16. An electric field created by a potential applied across electrode 14 and electrode 15 can provide another source of traverse force. In a favored mode, the device is operated with direct flow oriented against the gravity vector G. Separation medium that is relatively enriched in a given desired analyte (relative to an another analyte) can be withdrawn from one or more of the outlets 13.

In further illustration of such CFS devices, partial cross-section of CFS device 40 (shown in Figure 2) displays heat-exchange conduits 21 jacketing separation channel 30. Separation channel 30 is segmented by membranes 31.

CFS devices are typically of large scale, for example separation channels in excess of 300 millimeters in length are used to separate blood cell types. Design transformations and new operating methods allow these separations to be conducted in conveniently sized devices of 70 millimeters.

Summary of the Invention

In one embodiment, the invention relates to a separation device comprising: a separation channel having a width and height, the height defining a top and bottom of the channel, and a depth axis, the depth axis defining a first end and a second end of the channel, wherein the width is substantially greater than the height and the width is sufficient to provide effective lateral separation of analyte species; one or more liquid first inputs at the first end; two or more liquid first outputs at the second end; and one or more sources of traverse force comprising one or both of: (i) one or more traversing liquid inputs and one or more traversing liquid outputs, the traversing inputs and outputs adapted to provide liquid flow along an axis intersecting flow from the first inputs to the second inputs; and (ii) at least a pair of electrodes providing an electrical field across the width of the channel; wherein the device further comprises or is defined by: (a) two or more traversing liquid inputs and two or more traversing liquid outputs; or (b) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through a substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel; or (c) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through the substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel, wherein the openings are interior to the electrodes; or (d) slots on the top and bottom surfaces of the channel, and substantially parallel to the depth axis, into which slots are fitted diffusion-limiting meshes; or (e) a folded mesh fitted into the channel such that the spaces between the folds define included channels running substantially parallel to the depth axis; or (f) the absence of a heat exchange jacket contacting one or both of the top and bottom of the separation channel.

The invention also relates to a method of operating a separation device, comprising: (a) providing the device, which comprises: a separation channel; one or more liquid first inputs at the first end; one or more liquid first outputs at the second end, wherein the space of the separation channel between the first inputs and the first outputs defines a separation pathway; and one or more of traverse force elements comprising: (i) one or more traversing liquid inputs and one or more traversing liquid outputs, the traversing inputs and outputs adapted to provide liquid flow along an axis intersecting flow from the first inputs to the second inputs; or (ii) at least a pair of electrodes providing an electrical field across the width of the channel; (b) injecting sample containing one or more analytes into the separation channel; (c) providing direct liquid flow from the first inputs to the first outputs and coordinately operating one or more of the traverse force elements; (d) reversing analyte travel along the separation pathway by either: (i) stopping the direct liquid flow with the separation device oriented to provide settling of one or more of the analytes (and optionally also stopping one or more of the traverse flow elements); or (ii) reversing the direct liquid flow; and (e) after the reversing analyte travel step, again providing liquid flow across first inputs and the first outputs and coordinately operating one or more of the traverse force elements, wherein the amount and direction of settling or the amount of reverse direct flow is effective to increase the analyte separating effectiveness of the liquid flow providing steps. Brief Description of the Drawings

Figure 1 displays a top view of a typical CFS device.

Figure IB displays a cross section of a typical CFS device. Figure 2 displays a similar cross section of a typical CFS device.

Figure 3A displays a top view of the present invention.

Figure 3B displays a cross section of the present invention.

Figure 3C displays a similar cross-section of the present invention.

Figure 4 shows an embodiment where the membrane, or mesh, is folded in an accordion-like manner Detailed Description of the Invention

The device denoted as 60 in Figure 3A is a multi-compartment charge-flow separator according to an illustrative embodiment of the present invention. The charge-flow separation device 60 has a separation channel 61, made up of a series of subcompartments 63 defined by membranes (including meshes) 72. The membranes are substantially parallel to axis D, where "substantially" means favoring direct flow more than traverse (i.e., countercurrent) flow. Two electrodes 68, 69 are located on either end of the series of subcompartments. The illustrative dimensions shown in Figure 3 are in millimeters. The separation device comprises a series of first liquid inputs 62 at a first end 61A of the separation channel and first liquid outputs 64 at a second end. The separation device also comprises a series of first traversing liquid inputs 65 and second traversing inputs 70, as well as first traversing liquid outputs 66 and second traversing outputs 71. The space from first end 61 A to second end 61B defines a separation pathway.

Traversing liquid inputs 65, 70 can be fed by one or more pumps which preferably can individually meter liquid flow to the inputs. The pump(s) are preferably adapted to allow the flow rate to the traversing liquid inputs to be individually adjusted. Such adjustment can include, for example, creating a predetermined change in traverse (i.e., countercurrent) flow based on location along the axis of direct flow (for first end 61A to second end 61B). Similar pumps and flow rate adjustments can also be applied to the traversing liquid outputs 66, 71. In the illustrated embodiment, the device is formed from a first (top) body piece 60A secured to a second (bottom) body piece 60B by a screw or other fastening device. 67. Gasket 74 provides a fluid-tight seal. Note that the references to "top" and "bottom" are for convenience in describing the device, which in fact is favorably used in another orientation, as described elsewhere. The fastening devices can be used as electrical contact points for applying electrical potentials to the electrodes, and can incorporate electrical plugs or other connecting devices, as is known in the art.

Further illustrated in Figure 3B the membrane 72 is shown fitted into notches 73. The notches 73 can be, for example, machine cut or etched into CFS device 60. As illustrated, the membrane 72 is seated between a first (top) body piece 60A secured to a second (bottom) body piece 60B. In an alternative embodiment (Figure 4), the membrane, or mesh, is folded in an accordion-like manner into channel 161 of CFS device 160. The illustrative CFS device 160 is formed of a top body piece 160A, bottom body piece 160B and spacer body piece 160C. As illustrated, the device can be provided with retaining membranes 162. The membrane can be pre-folded in an accordion-like manner and inserted into channel, facilitating fitting into the channel. The membrane used can be, for example, woven polytetrafluroethylene, polyvinylidone fluoride, cellulose, nitrocellulose, polycarbonate, polysulfone, microporous glass or ceramics, or woven microfilament nylon screens.

Liquid separation medium can be inserted at inlets 62, and withdrawn at outlets 64, to provide "direct" flow into separation channel 61. The sample is inserted at an inlet selected as appropriate given the character of the separation to be obtained, as determined from test operations. Membrane/mesh 72 is designed to allow traverse fluid flow, and reduce convection currents and other mixing forces. One source of traverse force is provided by flow from traversing liquid inputs 65 to traversing liquid outputs 66. A potential applied across electrode 68 and electrode 69 can provide another source of traverse force.

In a favored mode, the device is operated with direct flow oriented against the gravity vector G. Separation medium that is relatively enriched in a given desired analyte (relative to an another analyte) can be withdrawn from one or more of the outlets 64. The device can be operated by injecting sample containing one or more analytes into the separation channel, providing direct liquid flow from the first inputs to the first outputs and coordinately operating one or more of the traverse force elements, i.e. traverse fluid flow or electrical potential.

In a preferred mode, direct liquid flow is stopped at intervals to allow settling of the analytes. Optionally, one or more of the traverse flow elements can also be stopped. The analytes settle to a point earlier in the separation pathway, while maintaining lateral separation. Each iteration of the stop flow protocol thereby adds to the effective length of the separation pathway. Alternatively, the separation pathway is lengthened by periodically reversing direct liquid flow.

The CFS devices of the invention can be formed of any suitable material that can be shaped to provide the separation channel, notches (if needed), inlets and outlets, and, if applicable, is of sufficient electrical resistivity to allow the applications of the electrode-generated fields. The shapes comprising the CFS device can be formed, for example, by compression molding, etching, machining, and the like. The segments of the inlets and outlets extending outside the body of the device can be individually shaped liquid connectors that fit into the device by, for example, compression fitting. Suitable materials for forming the device include moldable plastics (such as acrylic or polycarbonate), glass (such as temperature resistant borosilicate glass such as a Pyrex glass). If the CFS device is formed of separate layers, the layers can be sealed together with an appropriately shaped gasket, such as a gasket formed as described in US Patent 6,033,544, or a ring compression gasket (as illustrated) formed of an appropriate elastomer such as silicone or rubber. Layers of plastic can be sealed together by, for example, silicone, epoxy, or sonic welding. Note that second body piece 60B can itself be formed of two sub-pieces, with the channel segments of the traversing inlets or outlets illustrated as horizontal in Figure 3B formed on one or both joining faces of the sub-pieces, and the vertical channel segments formed in the upper sub- piece.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations in the preferred devices and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the claims that follow.

Claims

What is claimed:

1. A separation device comprising: a separation channel having a width and height, the height defining a top and bottom of the channel, and a depth axis, the depth axis defining a first end and a second end of the channel, wherein the width is substantially greater than the height and the width is sufficient to provide effective lateral separation of analyte species; one or more liquid first inputs at the first end; two or more liquid first outputs at the second end; and one or more sources of traverse force comprising one or both of: one or more traversing liquid inputs and one or more traversing liquid outputs, the traversing inputs and outputs adapted to provide liquid flow along an axis intersecting flow from the first inputs to the second inputs; and at least a pair of electrodes providing an electrical field across the width of the channel; wherein the device further comprises or is defined by:

(a) two or more traversing liquid inputs and two or more traversing liquid outputs; or

(b) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through a substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel; or

(c) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through the substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel, wherein the openings are interior to the electrodes; or

(d) slots on the top and bottom surfaces of the channel, and substantially parallel to the depth axis, into which slots are fitted diffusion-limiting meshes; or

(e) a folded mesh fitted into the channel such that the spaces between the folds define included channels running substantially parallel to the depth axis; or

(f) the absence of a heat exchange jacket contacting one or both of the top and bottom of the separation channel.

2. A separation device according to claim 1, comprising (a) two or more traversing liquid inputs and two or more traversing liquid outputs.

3. A separation device according to claim 1, further comprising: (g) one or more pumps adapted to meter liquid flow to traversing liquid inputs.

4. A separation device according to claim 3, wherein the pump(s) are adapted to allow the flow rate to the traversing liquid inputs to be individually adjusted.

5. A separation device according to claim 1, comprising (b) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through a substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel.

6. A separation device according to claim 1, comprising (c) two or more traversing liquid inputs and two or more traversing liquid outputs, wherein these inputs and outputs comprise channels through the substrate in which the separation channel is formed, the channels opening into the channel on a top or bottom surface of the channel, wherein the openings are interior to the electrodes.

7. A separation device according to claim 1, comprising (d) slots on the top and bottom surfaces of the channel, and substantially parallel to the depth axis, into which slots are fitted diffusion-limiting meshes.

8. A separation device according to claim 1, comprising (e) a folded mesh fitted into the channel such that the spaces between the folds define included channels running substantially parallel to the depth axis.

9. The mesh according to claim 8, wherein the mesh is pre-folded in an accordion-like manner and fitted into the channel.

10. A separation device according to claim 1, wherein the device is defined by (f) the absence of a heat exchange jacket contacting one or both of the top and bottom of the separation channel.

11. A method of operating a separation device, comprising: providing the device, which comprises: a separation channel; one or more liquid first inputs at the first end; one or more liquid first outputs at the second end, wherein the space of the separation channel between the first inputs and the first outputs defines a separation pathway; and one or more of traverse force elements comprising: one or more traversing liquid inputs and one or more traversing liquid outputs, the traversing inputs and outputs adapted to provide liquid flow along an axis intersecting flow from the first inputs to the second inputs; or at least a pair of electrodes providing an electrical field across the width of the channel, injecting sample containing one or more analytes into the separation channel; providing direct liquid flow from the first inputs to the first outputs and coordinately operating one or more of the traverse force elements; reversing analyte travel along the separation pathway by either: stopping the direct liquid flow with the separation device oriented to provide settling of one or more of the analytes; or reversing the direct liquid flow; and after the reversing analyte travel step, again providing liquid flow across first inputs and the first outputs and coordinately operating one or more of the traverse force elements, wherein the amount and direction of settling or the amount of reverse direct flow is effective to increase the analyte separating effectiveness of the liquid flow providing steps.

12. A method of operating a separation device according to claim 11, wherein the reversing analyte travel step comprises stopping the direct liquid flow with the separation device oriented to provide settling of one or more of the analytes.

13. The method of claim 11, further comprising also stopping one or more of the traverse flow elements in conjunction with the reversing analyte travel step.

14. A method of operating a separation device according to claim 11, wherein the reversing analyte travel step comprises reversing the direct liquid flow.