(12) United States Patent
`Wada et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,506,609 B1
`Jan. 14, 2003
`
`U5006506609Bl
`
`(54)
`
`(75)
`
`FOCUSING ()F MICROPARTICLES IN
`MICROFLUIDIC SYSTEMS
`
`Inventors: H. Garrett Wilda, Alherton, CA (US);
`Anne R. Kopf-Slll, Portola Valley, CA
`(US); Marja Liisa Alajoki, Palo Alto,
`CA (US); J. Wallace Parce, Palo Alto,
`CA (US); Benjamin N. Wang, Palo
`Alto, CA (US); Andrea W. Chow, Los
`Altos, CA (US); Robert S. Dubrow,
`San Carlos, CA (US)
`
`(73)
`
`Assignee:
`
`Caliper Technologies Corp., Mountain
`View. CA (US)
`
`(")
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21)
`
`(32)
`
`(60)
`
`Appl. No.:
`Filed:
`
`09/569,747
`
`May 1], 2000
`
`Related U.S. Application Data
`Provisional application No. 60/134,472, filed on May 17,
`1999.
`
`Int. Cl.7 .................................................. G01N 7/00
`U.S. CI.
`........................... 436/148; 436/34; 436/52;
`436/180; 436/518; 422/50; 435/911
`Field of Search ............................ 436/148, 34, 52,
`436/ 180, 518; 422/50; 204/452, 454, 600;
`356/73; 435/71, 6, 287.3, 91.1; 210/634
`
`356/73
`
`5,593,838 A
`5,603,351 A
`5,618,519 A
`5 635,358 A
`5,637,469 A
`5,699,157 A
`5,739,902 A *
`5,750,015 A
`5,779,868 A
`5,800,690 A
`5,842,787 A
`
`1/1997 [anzucchi et al.
`2/1997 Cherukuri ct al.
`3/1997 (jourley et al.
`6/1997 Wilding et al.
`6/1997 Wilding et al.
`12/1997 Parcc
`4/1998 (jjelsnes el al.
`5/1998 Soanc ct al.
`7/1998 Farce et al.
`9/1998 Chow et al.
`12/1998 Kopf—Sill et al.
`
`(List continued on next page.)
`FOREIGN PATENT DOCUMENTS
`
`W0
`W0
`W0
`W0
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`W0 96/114547
`WO 97/02357
`WO 98/00231
`WO 98/00705
`
`2/‘1996
`1/1997
`1/1998
`1/1998
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`OTHER PUBLICATIONS
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`Cohen, CB. ct al., “A Microchip—Based Enzyme Assay for
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`Dasgupta, P.K., et al. “Electroosmosis: A Reliable Fluid
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`Chem. (1994) 66:1792—1798.
`
`(List continued on next page.)
`
`Primary Examiner—Jill Warden
`Assistant Examiner—Brian Sines
`(74) Attorney, Agent. or Firm—Andrew L. Filler
`
`(57)
`
`ABS'I RACT
`
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`
`35 Claims, 22 Drawing Sheets
`
`
`
`ABS Global, Inc. and Genus pic — Ex. 1006, p. 1
`
`

`

`US 6,506,609 B1
`
`Page 2
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`2047452
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`W0
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`wo
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`l ,
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`,, P} ’
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`Lonlrol of Mud Flow Wilhm a Manifold of Capillaries on
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`‘ ciled by examiner
`
`ABS Global, Inc. and Genus plc – Ex. 1006, p. 2
`ABS Global, Inc. and Genus plc — EX. 1006, p. 2
`
`

`

`US. Patent
`
`Jan. 14, 2003
`
`Sheet 1 of 22
`
`US 6,506,609 B1
`
`1 {34-
`
`Fig. 1A
`
`
`
`Fig, 23
`
`ABS Global, Inc. and Genus plc — Ex. 1006, p. 3
`
`

`

`US. Patent
`
`Jan. 14, 2003
`
`Sheet 2 of 22
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`US 6,506,609 B1
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`

`U.S. Patent
`
`Jan. 14, 2003
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`Sheet 3 0f 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 5
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`

`U.S. Patent
`
`Jan. 14, 2003
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`US 6,506,609 B1
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`US. Patent
`
`Jan. 14, 2003
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 7
`ABS Global, Inc. and Genus plc — EX. 1006, p. 7
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`

`

`US. Patent
`
`Jan. 14, 2003
`
`Sheet 6 or 22
`
`US 6,506,609 B1
`
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`U.S. Patent
`
`Jan. 14, 2003
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`Sheet 7 of 22
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`US 6,506,609 B1
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`U.S. Patent
`
`Jan. 14, 2003
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`

`US. Patent
`
`Jan. 14, 2003
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`Sheet 10 of 22
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`Us 6,506,609 B1
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 12
`ABS Global, Inc. and Genus plc — EX. 1006, p. 12
`
`

`

`U.S. Patent
`
`Jan. 14, 2003
`
`Sheet 11 of 22
`
`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 13
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`

`

`US. Patent
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`Jan. 14, 2003
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`Sheet 12 0f 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 14
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`

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`US. Patent
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`Jan. 14, 2003
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`Sheet 13 0f 22
`
`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — EX. 1006, p. 15
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`

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`US. Patent
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`Jan. 14, 2003
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`Sheet 14 of 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 16
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`

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`US. Patent
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`Jan. 14,2003
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`Sheet 15 0f 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 17
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`US. Patent
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`Jan. 14, 2003
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`Sheet 16 0f 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 18
`ABS Global, Inc. and Genus plc — EX. 1006, p. 18
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`US. Patent
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`Jan. 14, 2003
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`Sheet 17 of 22
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`US. Patent
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`Jan. 14, 2003
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`Sheet 18 of 22
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`ABS Global, Inc. and Genus plc — EX. 1006, p. 20
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`US. Patent
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`Jan. 14, 2003
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`Sheet 19 0f 22
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`US 6,506,609 B1
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`US. Patent
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`Jan. 14,2003
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`Sheet 20 0f 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 22
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`US. Patent
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`Jan. 14,2003
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`Sheet 21 0f 22
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`US 6,506,609 B1
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`Fluorescencce Intensity (a. u )
`
`Fig.258
`
`ABS Global, Inc. and Genus plc — Ex. 1006, p. 23
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`

`

`US. Patent
`
`Jan. 14, 2003
`
`Sheet 22 of 22
`
`US 6,506,609 B1
`
`Frequency
`
`Q"
`9‘
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`
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`
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`
`Fluorescence Signal
`
`Fig. 26
`
`ABS Global, Inc. and Genus plc — Ex. 1006, p. 24
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`

`

`US 6,506,609 B1
`
`1
`FOCUSING 0F MICROPARTICLES IN
`MICR()FI..UII)IC SYSTEMS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is related to and claims priority to and the
`benefit of provisional application 60l134,472, filed May 17,
`1999, Wada et al., "Focusing of Microparticles in Microf-
`luidic Systems,” pursuant to 35 U.S.C. §119(e), as well as
`any other applicable statute or rule. This priority application
`is incorporated herein in its entirety for all purposes.
`
`COPYRIGHT NOTIFICATION
`
`to 37 C.F.R. 1.?1(e), Applicants note that a
`Pursuant
`portion of this disclosure contains material which is subject
`to copyright protection. The copyright owner has no objec-
`tion to the facsimile reproduction by anyone of the patent
`document or patent disclosure, as it appears in the Patent and
`Trademark Oflice patent
`file or records, but otherwise
`reserves all copyright rights whatsoever.
`
`BACKGROUND OF THE INVENTION
`
`A variety of cell-based assays are of considerable com-
`mercial relevance in screening for modulators of cell-based
`activity. For example, compounds which affect cell death
`can have profound biological activities and are desirably
`screened for in cell-based assays. Cell death has become
`recognized as a physiological process important in normal
`development, hormonal regulation of various tissues, and,
`e.g., in regulation of the receptor repertoires of both 'I' and
`B lymphocytes. The finding that a pattern of morphological
`changes is common to many examples of programmed cell
`death (or
`I’CD)
`led to the suggestion of a common
`mechanism. and the term "apoptosis” was defined to include
`both the morphological features and the mechanism com-
`mon to such programmed cell death (Kerr et al., Bi: J.
`Cancer 26:239). This concept was extended by the finding
`that nuclear DNA fragmentation correlates well with apop-
`totic morphology (Arends et al., Am. J. Partial. 136:593
`(1990)), and the scientific literature contains many examples
`of PCD accompanied by these features. There are also clear
`examples of PCD in the absence of apoptotic morphology or
`DNA fragmentation (Clarke,Annr. Embryl. 181: 195 (1990),
`Martin et at, J. CettBiot. 106:829 (1988), and Ishigami et al.,
`J'. Innmmof. 1482360 (1992)).
`relevant biological
`Cell-based assay systems model
`phenomena, and have generally been widely adopted as
`screening assays, e.g., when screening for a compound’s
`elfect(s) on apoptosis or other biological phenomena. Pio-
`neering technology providing cell— and other particle—based
`microscale assays are set
`forth in Parce et at “High
`Throughput Screening Assay Systems in Microscale Fluidic
`Devices” W0 98r’00231; in PCTfUSfl0104522, tiled Feb. 22,
`2000, entitled “Manipulation of Micropartictes tn Microf-
`tuidic Systems," by Mehta et al., and in PCTUStXIIU4486,
`tiled Feb. 22, 2000, entitled “Devices and Systems for
`Sequencing by Synthesis,“ by Mehta et at.
`Other cell—based assays include various methods for the
`preparative or analytic sorting ofdifferent types of cells. For
`example, cell panning generally involves attaching an
`appropriate antibody or other cell—specific reagent to a solid
`support and then exposing the solid support to a heteroge-
`neous cell sample. Cells possessing, e.g., the corresponding
`membrane-bound antigen will bind to the support, leaving
`those lacking the appropriate antigenic determinant to be
`
`it]
`
`15
`
`3o
`
`35
`
`40
`
`45
`
`50
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`55
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`60
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`65
`
`2
`washed away. Other well-known sorting methods include
`those using fluorescence-activated cell sorters (“I“ACSs“).
`FACSs for use in sorting cells and certain suhccllular
`components such as molecules of DNA have been proposed
`in. e.g., Fu, A. Y. et
`a].
`(1999) “A Microfabricated
`Fluorescence-Activated Cell Sorter," Not. Biotechrtol.
`17:1109—1111; Unger, M., et a1. (1999) “Single Molecule
`Fluorescence Observed with Mercury Lamp Iluminalion,"
`Biotect‘mr’ques 271008—1013; and Chou, It. 1’. et at. (19.99)
`“A Microt'abricated Device for Sizing and Sorting DNA
`Molecules," Pmc. Nat "t. Acad. Sci. 96: 1 1—13. These sorting
`techniques utilizing generally involve focusing cells or other
`particles by flow channel geometry.
`While cell-based assays are generally preferred in certain
`microscale screening applications, certain of these assays
`are dillicult
`to adapt
`to conventional notions of high-
`throughput or ultra high-throughput screening assay sysv
`terns. For example, one difficulty in flowing assay systems
`is that, during pressure-based flow of” fluids in channels,
`non-uniform flow velocities are experienced. Faster fluid
`and material flow is observed in the center of a moving fluid
`stream than on the edge of a moving fluid stream. This
`non-uniform flow velocity reduces throughput for flowing
`assays, because assay runs have to be spaced well apart in
`the fluid stream to prevent overlap of materials moving at
`dilIerent velocities.
`
`Accordingly, it would be advantageous to provide mecha-
`nisms for facilitating cell-based assays, including cell sort-
`ing techniques, especially in microscale systems. Additional
`microscale assays directed at subcetlular components, such
`as nucleic acids would also be desirable. The present inven-
`tion provides these and other features which will become
`clear upon consideration of the following.
`
`SUMMARY OF THE INVENTION
`
`invention relates to methods of focusing
`The present
`particles in microchannels, e.g.,
`to improve assay
`throughput, to sort particles, to count particles, or the like. In
`the methods 01" the invention, cells and other particles are
`focused in the center of, to one side of, or in other selected
`regions of microscale channels, thereby avoiding, e.g., the
`above noted difficulties inherent
`in pressure—based flow of
`particles. Furthermore, the device structures of the present
`invention are optionally integrated with other microfluidic
`systems. Other reactions or manipulations involving cells,
`other particles, or fluids upstream of the detection zone are
`also optionally performed, e.g., monitoring drug interactions
`with cells or other particles.
`In one aspect, the invention provides methods of provid-
`ing substantially uniform flow velocity to particles flowing
`in a first microchannel.
`tn the methods. the particles are
`optionally flowed in the microchannel, e.g., using pressure-
`based flow, in which the particles flow with a substantially
`non-uniform flow velocity. Prior to performing the flowing
`step, the particles are optionally sampled with at least one
`capillary element, e.g., by dipping the capillary element into
`a well containing the particles on a microwell plate and
`drawing the particles into, e.g., reservoirs, microchannels, or
`other chambers of the device. The particles (e.g., a cell. a set
`of cells, a microbead, a set of microbeads, a functionalized
`mierobead, a set of t‘u nctionalized microbeads, a molecule,
`a set of molecules, etc.) are optionally focused horizontally
`andi'or vertically in the first microchannel to provide sub-
`stantially uniform flow velocity to the particles in the first
`microchannel. Particles are optionally focused using one or
`more fluid direction components {e.g., a fluid pressure force
`
`ABS Global, Inc. and Genus plc – Ex. 1006, p. 25
`ABS Global, Inc. and Genus plc — EX. 1006, p. 25
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`

`

`US 6,506,609 B1
`
`3
`modulator an electrokinetic force modulator, a capillary
`force modulator, a fluid wicking element, or the like).
`Additional options include sorting, detecting or otherwise
`manipulating the focused particles.
`The particles are horizontally focused in the
`microchannel, e.g., by introducing a low density fluid and a
`high density fluid into the microchannel, causing the par-
`ticles to he focused in an intermediate density fluid present
`between the high density fluid and the low density fluid. The
`particles are also optionally focused in a top or a bottom
`portion of the microchannel by introducing a high or a low
`density fluid into the microchannel with the flowing par-
`ticles. The particles are vertically or horizontally focused in
`the microchannel, e.g., by simultaneously introducing fluid
`flow from two opposing microchannels into the first micro—
`channel during flow of the particles in the first channel.
`Vertical focusing is also optionally achieved to one side of
`a microchannel by simultaneously introducing fluid flow
`from, e.g., a second microchannel into the first microchannel
`during flow of the particles in the first microchannel.
`In another aspect,
`the invention also provides particle
`washing or exchange techniques. For example, foeused cells
`or other particles are optionally washed free of dilIusible
`material by introducing a diluent into the lirst microchannel
`from at least a second channel and removing the resulting *
`diluted difl'used product comprising diluent mixed with the
`dilIusible material through at least a third microchannel.
`Alternating arrangements of diluent
`input and difl'used
`product output channels are also optionally used to further
`wash the particles. For example, in one aspect the methods
`of the invention include simultaneously introducing the
`diluent into the first microchannel from the second miero-
`channel and a fourth microchannel, where the second and
`fourth microchannel intersect
`the first microchannel at a
`common intersection region. Optionally,
`the methods
`include sequentially introducing the diluent
`into the first
`microchannel from the second microchannel and a fourth
`microchannel, wherein the second and fourth microchannels
`intersect
`the first microchannel at an offset
`intersection
`region. The dilIused product is typically removed through
`the third microchannel and a fifth microchannel, which third
`and fifth microchannels intersect the first microchannel at a
`common intersection region. In further washing steps, the
`diluent is introduced through sixth and seventh microchan-
`ncls which intersect
`the first microchannel at a common
`intersection. The resulting further diluted diffused product is
`removed through eighth and ninth mierochannels, which
`intersect the first microchannel at a common intersection.
`Diluent is optionally introduced into the first microchannel
`by pressure or electrokinetic flow.
`In one preferred assay of the invention, the particles are
`cells and the method includes performing a TUNEL assay or
`an Annexin-V assay on the cells in the channel to measure
`apoptosis.
`Integrated systems for performing the above methods,
`including the particle sorting embodiments, are also pro-
`vided.
`
`it]
`
`15
`
`so
`
`35
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`40
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`50
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`
`4
`generally induces non-uniform flow. Asource of at least one
`lluidic material
`is optionally fluidly coupled to the first
`microchannel. The system also optionally includes at least a
`second microchannel that intersects the first microchannel
`
`for introducing at least one fluid into the first microchannel
`to horizontally or vertically focus the members ofthe at least
`one particle population in the first microchannel. The at least
`one fluid is optionally introduced using a second fluid
`direction component that includes one or more of a fluid
`pressure force modulator, an electrokinetic force modulator,
`a capillary force modulator, a fluid wicking element, or the
`like. At least one flow control regulator for regulating flow
`of the fluidic material or the fluid in the first or second
`microchannel
`is also optionally provided. A computer
`including an instruction set directing simultaneous flow of
`the members of the at least one particle population in the first
`microchannel and simultaneous introduction of the at least
`one fluid from the second microchannel into the first micro-
`channel is optionally also operahly coupled to a fluid move-
`ment system for directing flow of materials in the micro-
`channels.
`As a further option, this integrated system additionally
`includes at least a third microchannel which intersects the
`first microchannel in an intersection region common to the
`second microchannel. The flow control regulator of this
`system optionally further regulates flow of the at least one
`fluid in the second and the third microchannels.
`In this
`embodiment,
`the computer typically also includes an
`instruction set for simultaneously flowing lluids from the
`second and third microchannels into the first microchannel.
`
`In particle washing systems, typically, at least fourth and
`fifth channels which intersect the first microchannel at a
`common intersection downstream of the second and third
`microchannels are provided. The computer further includes
`an instruction set for simultaneously flowing material from
`the first microchannel into the fourth and fifth microchan-
`nels. Sixth and seventh microchannels which intersect the
`first microchannel at a common intersection downstream of
`the fourth and fifth mierochannels, with the computer further
`comprising an instniction set for simultaneously flowing
`material from the sixth and seventh microchannels into the
`first microchannel are optionally provided. Similarly, eighth
`and ninth microchannels which intersect the first microchan-
`nel at a common intersection downstream of the sixth and
`seventh mierochannels, the computer further including an
`instniction set for simultaneously flowing material from the
`first microchannel into the eighth and ninth microchannels
`are optionally provided.
`The integrated system optionally includes sources for any
`reagent or particle used in the methods noted above, such as
`one or more sources of terminal deoxynucleotide
`transferase, one or more sources of one or more lluoresoein
`labeled nucleotides or other labeled polynucleotides, one or
`more sources of Annexin V, one or more sources of an
`AnnexinV—biotin conjugate, one or more sources of a DNA
`dye, one or more sources of Campthotecin, one or more
`sources of Calcein-AM, one or more sources of a control
`cell, one or more sources of a test cell, etc.
`Signal detector(s) mounted proximal to the first micro-
`channel for detecting a detectable signal produced by one or
`more of the members of the at least one particle population
`in the microchannel are typically provided in the integrated
`systems of the invention. The detector also optionally
`includes, e.g., a fluorescent excitation source and a fluores—
`cent emission deteetion element. Optionally, the computer is
`operably linked to the signal detector and has an instruction
`set for converting detected signal information into digital
`data.
`
`An integrated system for providing substantially uniform
`flow velocity to flowing members of at
`least one particle
`population in a microfluidic device optionally includes a
`body structure that includes at
`least a first microchannel
`disposed therein. A first fluid direction component (e.g,, a
`fluid pressure force modulator) is typically coupled to the
`first microchannel for inducing flow of a fluidic material that
`includes the members of the at least one particle population
`in the first microchannel. The first fluid direction component
`
`60
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 26
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`US 6,506,609 B1
`
`5
`invention is also
`The integrated system of the present
`optionally used to sort the members of a particle population
`(e.g., a cell, a set of cells, a microbead, a set of microbcads,
`a functionalized microbead, a set of functionalized microbe
`ads, a molecule, a set of molecules, or the like). In this
`embodiment,
`the integrated system typically additionally
`includes a third and a fourth microchannel which intersect
`the first microchannel downstream from the intersection of
`the second microchanne] with the first microchannel. The
`fourth microchannel also generally intersects the first micro-
`channel downstream from the intersection of the third
`microchannel with the first microchannel. The flow control
`regulator of this system optionally further regulates flow of
`the at least one fluid in the third or the fourth microchannels.
`Furthermore, the signal detector typically detects a detect-
`able signal produced by a selected member of the particle
`population between the intersections of the second and the
`third microchannels with the first microchannel.
`
`in
`
`15
`
`-
`
`the computer is
`In this particle sorting embodiment,
`optionally operably linked to the first or other fluid direction
`component(s),
`the flow control regulator, and the signal
`detector. Additionally, the instruction set
`typically directs
`simultaneous introduction of the at least one fluid from the
`third microehannel into the first microchannel to horizon-
`tally or vertically focus the selected member of the particle
`population such that the selected member is directed into the _
`fourth microchannel
`in response to the detectable signal
`produced by the selected member. Optionally, the instruction
`set further directs simultaneous introduction of the at least
`one fluid from the third microchannel by activating a heating
`element (e.g., a Joule heating electrode, a conductively
`coated microchannel portion, etc.) disposed within the third
`microchannel or a well that fluidly communicates with the
`third microehannel.
`
`3o
`
`least a portion of the first
`In another embodiment, at
`microchannel optionally includes a separation element dis-
`posed therein. The separation element optionally includes,
`e.g., two sides and at
`least a portion of the separation
`element is typically disposed upstream of the fourth micro-
`channel.
`In this embodiment, a selected member of the
`particle population is generally directed to one of the two
`sides of the separation element and into the fourth micro-
`channel that intersects the first microchannel in response to
`the detectable signal produced by the selected member.
`The integrated system for use in particle sorting also
`optionally includes a tiflh microchannel which intersects the
`first microchannel in an intersection region common to the
`seconrl microchannel. In this case, the flow control regulator
`also typically regulates flow of the at least one fluid in the
`second and the tiflh microchannels, and the computer
`optionally includes an instruction set for simultaneously
`flowing fluids from the second and the fifth mierochannels
`into the first microchannel. Similarly,
`the system also
`optionally includes a sixth mierochannel which intersects
`the first microchannel in an intersection region common to
`the third microchannel. In this embodiment, the flow control
`regulator optionally additionally regulates flow of the at
`least one fluid in the third and the sixth micro-channels.
`Furthermore.
`the computer also typically includes an
`instruction set for flowing fluids from the third and the sixth
`microchannels into the first microchannet. Optionally, the
`instruction set directs individual or simultaneous fluid flow
`from the third and sixth mierochannels by individually or
`simultaneously activating at least one heating element (e.g.,
`a Joule heating electrode, a conductivcly coated microchan—
`nel portion, or the like) disposed within each of the third and
`Sixth microehanneLs or within at least one well that fluidly
`communicates with each of the third and sixth microchan-
`nets.
`
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`6
`Many additional aspects of the invention will be apparent
`upon review, including uses of the devices and systems of
`the invention, methods of manufacture of the devices and
`systems of the invention, kits for practicing the methods of
`the invention and the like. For example. kits comprising any
`of the devices or systems set forth above, or elements
`thereof,
`in conjunction with packaging materials (cg,
`containers, sealable plastic bags, etc.) and instructions for
`using the devices, e.g., to practice the methods herein, are
`also contemplated.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1A is a schematic showing focusing of cells in a
`microscale system by simultaneous flow from side channels
`into a main channel
`through which the cells are being
`flowed. FIG. 1B is a photomicrograph of focused labeled
`cells flowing in a microchannel.
`FIG. 2A is a schematic of a microfluidic system with a
`pressure-source {in this case a vacuum source} for achieving
`fluid movement. FIG. 2B shows a cross-sectional view down
`
`a channel having high, medium and low fluid density.
`FIG. 3 is a schematic drawing of a microfluidic system
`adapted to washing reagents from microparticles.
`FIG. 4 is a data graph illustrating a control analysis, e.g.,
`U937 cells not treated to induce apoptosis. The bottom line
`corresponds to the SYTO®-62, which indicates the mere
`presence of cells, whereas the top line corresponds to the
`fluorescein end labeled nucleic acids.
`
`FIG. 5 is a data graph illustrating U937 cells treated with
`Campthotecin to induce apoptosis. As can be seen, corre—
`sponding peaks are seen on both the lower and upper lines,
`indicating the presence of apoptotic cells.
`FIG. 6 is a data graph illustrating an analysis ofU937 cells
`that were not treated to induce apoptosis. In thi