(12)
`
`United States Patent
`Wadaet al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,506,609 B1
`Jan. 14, 2003
`
`US006506609B1
`
`(54)
`
`(75)
`
`(73)
`
`FOCUSING OF MICROPARTICLESIN
`MICROFLUIDIC SYSTEMS
`
`Inventors: H. Garrett Wada, Atherton, CA (US);
`Anne R. Kopf-Sill, 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)
`
`Assignee: Caliper Technologies Corp., Mountain
`View, CA (US)
`
`5,593,838 A
`5,603,351 A
`5,608,519 A
`5,635,358 A
`5,637,469 A
`§,699,157 A
`5,739,902 A *
`5,750,015 A
`5,779,868 A
`5,800,690 A
`A
`5,842,787
`
`1/1997 Zanzucchiet al.
`2/1997 Cherukuri et al.
`3/1997 Gourleyet al.
`6/1997 Wilding et al.
`6/1997 Wilding etal.
`12/1997 Parce
`4/1998 Gjelsnes et al.
`5/1998 Soaneet al.
`7/1998 Parce et al.
`9/1998 Chowetal.
`12/1998 Kopf-Sill et al.
`
`.............. 356/73
`
`(List continued on next page.)
`FOREIGN PATENT DOCUMENTS
`
`wo
`WO 96/04547
`2/1996
`Wo
`WO 97/02357
`1/1997
`e*)
`Notice:|Subject to any disclaimer, the term of this
`WO
`WO 98/00231
`1/1998
`patent is extended or adjusted under 35
`Wo
`WO 98/00705
`1/1998
`U.S.C. 154(b) by 0 days.
`
`(21)
`
`(22)
`
`(60)
`
`Appl. No.: 09/569,747
`
`Filed:
`
`May11, 2000
`
`Related U.S. Application Data
`Provisional application No. 60/134,472, filed on May 17,
`1999.
`
`Wait O02?szcssnsescrsea.uaannuescnaseveansciniaeates GOIN 7/00
`USC sicssssnscorsnseverssszaseans 436/148; 436/34; 436/52;
`436/180; 436/518; 422/50; 435/911
`Field of Search ............ccceseeee 436/148, 34, 52,
`436/180, 518; 422/50; 204/452, 454, 600;
`356/73; 435/7.1, 6, 287.3, 91.1; 210/634
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
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`(List continued on next page.)
`
`Primary Examiner—Jill Warden
`Assistant Examiner—Brian Sines
`(74) Attorney, Agent, or Firm—Andrew L. Filler
`
`(57)
`
`ABSTRACT
`
`Methods and systemsfor particle focusing to increase assay
`throughput in microscale systems are provided. The inven-
`tion includes methods for providing substantially uniform
`flow velocity to flowing particles in microfluidic devices.
`Methodsofsorting members ofparticle populations, such as
`cells and various subcellular components are also provided.
`Integrated systems in which particles are focused and/or
`sorted are additionally included.
`
`35 Claims, 22 Drawing Sheets
`
`ABSGlobal, Inc. and Genus ple — Ex. 1006,p. 1
`
`

`

`US 6,506,609 B1
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`Page 2
`
`U.S. PATENT DOCUMENTS
`
`............. 204/452
`
`............ 422/50
`
`wo
`wo
`wo
`wo
`wo
`wo
`Wo
`wo
`wo
`wo
`wo
`wo
`wo
`
`WO 98/46438
`WO 98/49548
`WO 98/55852
`WO 98/56956
`WO 99/00649
`WO 99/10735
`WO99/12016
`WO 99/16162
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`WO 99/19516
`WO 99/29497
`WO 99/56954
`WO 00/09753
`
`10/1998
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`;
`;
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`chip Electrophoresis,” Anal. Chem. (1995) 67:2059-2063.
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`5,965,410 A
`Knight J., et al., “Hydrodynamic Focusing on a Silien Chip:
`LOHS99 Parce ctal.
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`sce
`peice
`:
`7H
`oa
`i
`5,972,622 A * 10/1999 Desjardins .........2... 435/71
`Mixing Nanoliters in Microseconds” Physical Review Let-
`5.972.710 A
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`fers (1998) vol. 80,No. 17 pp. 3863-3866.
`;
`5,976,336 A
`11/1999 Dubrow et al.
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`5,989,402 A
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`pler anemometric analysis of particel mixtures in a channel
`6,001,231 A * 12/1999 Kopf-Sill ........002 204/454
`flow...” J. of Chromatography (1991) vol. 553 pp.
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`6,012,902 A
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`phoretic separations for miniaturized chemical analysis sys-
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`:
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`ment systems,” Nature Med. (1995) 1:1093-1096,
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`Seiler, K. et al., “Planar Glass Chips for Capillary Electro-
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`phoresis: Repetitive Sample Injection, Quantitation, and
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`Separation Efficiency,” Anal. Chem. (1993) 65:1481-1488.
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`a Glass Chip,” Anal. Chem. (1994) 66:3485-3491.
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`pul screening: solution—and cell-based approches,” Cur-
`rent Opinions in Biotechnology 2000, 11:47-53.
`Watson, J. “The Early Fluidic and Optical Physics of Cytom-
`etry” Cytometry (1999) vol. 38 pp. 2-14.
`
`wo
`wo
`wo
`
`wo
`wo
`
`WO 98/00707
`WO 98/02728
`WO 98/05424
`WO 98/22811
`WO 98/45481
`WO98/45929
`
`1/1998
`1/1998
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`10/1998
`
`* cited by examiner
`
`ABS Global, Inc. and Genus plc – Ex. 1006, p. 2
`ABS Global, Inc. and Genusplc — Ex. 1006, p. 2
`
`

`

`U.S. Patent
`
`Jan. 14, 2003
`
`Sheet 1 of 22
`
`US 6,506,609 BI
`
`104
`
`
`Fig. 1A
`
`Fig. 1B
`
`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 3
`
`

`

`U.S. Patent
`
`Jan. 14, 2003
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`US 6,506,609 B1
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`202
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`206
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`208
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`210
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`212
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`204
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`200
`
`Fig. 2A
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`214
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`216
`
`Fig. 2B
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`ABS Global, Inc. and Genus ple — Ex. 1006, p. 4
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`Jan. 14, 2003
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`Sheet 3 of 22
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`U.S. Patent
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`US 6,506,609 BL
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 5
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`

`

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`
`US 6,506,609 Bl
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`
`Jan. 14, 2003
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`ABSGlobal, Inc. and Genusple — Ex. 1006,p. 6
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`

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`U.S. Patent
`
`Jan. 14, 2003
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`Sheet 5 of 22
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`US 6,506,609 B1
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`Seconds
`
`Fig..5
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`2905
`291029152920292529302935294029452950
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 7
`ABS Global, Inc. and Genusplc — Ex. 1006, p. 7
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`US 6,506,609 BI
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`Seconds
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`Fig.6
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`90
`100105110115120125130135140145150
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`-2:| 33
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`95
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 8
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`Jan. 14, 2003
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`100
`110115120125130135
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`US 6,506,609 B1
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`Fig.7
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`105
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 9
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`US 6,506,609 B1
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`U.S. Patent
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`Jan. 14, 2003
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`ABS Global, Inc. and Genusple — Ex. 1006, p. 10
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`Jan. 14, 2003
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`Sheet 9 of 22
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`U.S. Patent
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`US 6,506,609 Bl
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`ABS Global, Inc. and Genusple — Ex. 1006, p. 11
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`U.S. 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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`HL60 Cells- 6 hr CPT
`
`26 Apoptotic (52%)
`24 Alive (48%)
`
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`
`HL60 Cells- 6 hr DMSO
`
`1 Apoptotic (2.6%)
`37 Alive (97.4%)
`
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`
`Fig. 10
`
`ABS Global, Inc. and Genus plc – Ex. 1006, p. 12
`ABS Global, Inc. and Genusplc — Ex. 1006, p. 12
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`U.S. Patent
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`Jan. 14, 2003
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`Sheet 11 of 22
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`US 6,506,609 BL
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`6 hr CPT :
`
`U937 Cells- TUNEL Assay
`
`4 hr CPT
`
`Treatment
`
`4 hr CPT
`
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`
`50
`
`40
`30
`
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`
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`
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`
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`
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`
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`
`
`Jurkat Cells- TUNEL Assay
`
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`
`6 hr CPT
`
`4 hr DMSO
`
`WL
`
`Treatment
`
`Fig. 11
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`ABSGlobal, Inc. and Genus ple — Ex. 1006,p. 13
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`US 6,506,609 Bl
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`4440
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`4460
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`4480
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`4500
`
`4520
`
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`
`Fig. 12
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`
`Fig. 13
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 14
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`200
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`Fig. 14
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`ABSGlobal, Inc. and Genusple — Ex. 1006, p. 15
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`US.
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`Patent
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`Jan. 14, 2003
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`Sheet 14 of 22
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`HL-60 Cells- Annexin-V Binding
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`
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`
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`
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`Fig 15
`
`ABSGlobal, Inc. and Genusple — Ex. 1006, p. 16
`
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`
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`
`
`
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`
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`Jan. 14, 2003
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`Sheet 15 of 22
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`US 6,506,609 B1
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`Fig. 17
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`ABS Global, Inc. and Genusple — Ex. 1006, p. 17
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`Jan. 14, 2003
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`Sheet 16 of 22
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`US 6,506,609 BI
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`ABS Global, Inc. and Genus plc – Ex. 1006, p. 18
`ABS Global, Inc. and Genusplc — Ex. 1006, p. 18
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`Sheet 18 of 22
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`US 6,506,609 BI
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`ABS Global, Inc. and Genus plc — Ex. 1006, p. 20
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`Sheet 19 of 22
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`US 6,506,609 B1
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`ABS Global, Inc. and Genusple — Ex. 1006, p. 21
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`2312
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`Fig. 24
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`ABS Global, Inc. and Genusple — Ex. 1006, p. 22
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`Sheet 21 of 22
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`US 6,506,609 B1
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 23
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`Jan. 14, 2003
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`Sheet 22 of 22
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`Fig. 26
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`ABSGlobal, Inc. and Genus ple — Ex. 1006, p. 24
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`US 6,506,609 B1
`
`1
`FOCUSING OF MICROPARTICLES IN
`MICROFLUIDIC SYSTEMS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This applicationis related to and claims priority to and the
`benefit of provisional application 60/134,472, 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. 1.71(e), Applicants note that a
`Pursuant
`portion ofthis disclosure contains material which is subject
`to copyright protection. The copyright owner has no objec-
`tion to the facsimile reproduction by anyone ofthe patent
`documentorpatent disclosure, as it appears in the Patent and
`Trademark Office 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 ofcell-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 T and
`B lymphocytes. The finding that a pattern of morphological
`changes is common to many examples of programmed cell
`death (or PCD)
`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., Br J.
`Cancer 26:239), This concept was extended by the finding
`that nuclear DNA fragmentation correlates well with apop-
`totic morphology (Arends et al., An. J. Pathol. 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
`DNAfragmentation (Clarke, Anat. Embryl. 181:195 (1990),
`Martin et al, J. Cel! Biol. 106:829 (1988), and Ishigami et al.,
`J. Immunol. 148:360 (1992)).
`relevant biological
`Cell-based assay systems model
`phenomena, and have generally been widely adopted as
`screening assays, ¢.g., when screening for a compound’s
`effect(s) on apoptosis or other biological phenomena. Pio-
`neering technology providing cell- and other particle-based
`microscale assays are set
`forth in Parce et al. “High
`Throughput Screening Assay Systems in Microscale Fluidic
`Devices” WO 98/00231; in PCT/US00/04522,filed Feb. 22,
`2000, entitled “Manipulation of Microparticles In Microf-
`luidic Systems,” by Mehta et al.; and in PCTUS00/04486,
`filed Feb. 22, 2000, entitled “Devices and Systems for
`Sequencing by Synthesis,” by Mehta etal.
`Other cell-based assays include various methods for the
`preparative or analytic sorting of different 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
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`washed away. Other well-known sorting methods include
`those using fluorescence-activated cell sorters (*FACSs”).
`FACSs for use in sorting cells and certain subcellular
`components such as molecules of DNA have been proposed
`in, e.g., Fu, A. Y. et al.
`(1999) “A Microfabricated
`Fluorescence-Activated Cell Sorter,” Nat. Biotechnol.
`17:1109-1111; Unger, M., et al. (1999) “Single Molecule
`Fluorescence Observed with Mercury Lamp [lumination,”
`Biotechniques 27:1008—1013; and Chou, H. P. et al. (1999)
`“A Microfabricated Device for Sizing and Sorting DNA
`Molecules,” Proc. Nat'l. Acad. Sci. 96:11-13. These sorting
`techniquesutilizing 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 difficult
`to adapt
`to conventional notions of high-
`throughput or ultra high-throughput screening assay sys-
`tems. 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
`different 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 subcellular 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 countparticles, or the like. In
`the methods of 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,
`otherparticles, 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.
`In the methods, the particles are
`optionally flowed in the microchannel, ¢.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
`drawingthe 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
`microbead, a set of functionalized microbeads, a molecule,
`a set of molecules, etc.) are optionally focused horizontally
`and/or vertically in the first microchannel to provide sub-
`stantially uniform flow velocity to the particles in thefirst
`microchannel. Particles are optionally focused using one or
`more fluid direction components (c.g., a fluid pressure force
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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 be focusedin 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 ofthe particles in the first microchannel.
`In another aspect,
`the invention also provides particle
`washing or exchange techniques. For example, focused cells
`or other particles are optionally washed free of diffusible
`material by introducing a diluent into the first microchannel
`from at least a second channel and removing the resulting
`diluted diffused product comprising diluent mixed with the
`diffusible material through at least a third microchannel.
`Alternating arrangements of diluent
`input and diffused
`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 micro-
`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 diffused product is typically removed through
`the third microchannel and a fifth microchannel, which third
`and fifth microchannelsintersect the first microchannel at a
`common intersection region. In further washing steps, the
`diluent is introduced through sixth and seventh microchan-
`nels which intersect
`the first microchannel at a common
`intersection. The resulting further diluted diffused productis
`removed through eighth and ninth microchannels, 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 TUNELassay 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.
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`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 fuid direction component (e.g., a
`fluid pressure force modulator) is typically coupled to the
`first microchannelfor inducing flow ofa fluidic material that
`includes the membersofthe at least one particle population
`in the first microchannel. Thefirst fluid direction component
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`generally induces non-uniform flow. A sourceof at least one
`fluidic material
`is optionally fuidly 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 fluidinto the first microchannel
`to horizontally or vertically focus the members ofthe atleast
`one particle populationin the first microchannel, Theat 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 low
`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 membersof the at least one particle population in the first
`microchannel and simultaneous introduction ofthe at least
`one fluid from the second microchannelinto the first micro-
`channel is optionally also operably 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 fluids 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 microchannels, with the computer further
`comprising, an instruction set for simultaneously flowing
`material from the sixth and seventh microchannels into the
`first microchannel are optionally provided. Similarly, eighth
`and ninth microchannels whichintersect the first microchan-
`nel at a common intersection downstream of the sixth and
`seventh microchannels, the computer further including an
`instruction 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 orparticle used in the methods noted above, such as
`one or more sources of terminal deoxynucleotide
`transferase, one or more sources of one or more fluorescein
`labeled nucleotides or other labeled polynucleotides, one or
`more sources of Annexin V, one or more sources of an
`Annexin V-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 ofa test cell, ete.
`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 detection element. Optionally, the computeris
`operably linked to the signal detector and has an instruction
`sel for converting detected signal information into digital
`data.
`
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`US 6,506,609 B1
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`5
`invention is also
`The integrated system of the present
`optionally used to sort the members ofa particle population
`(e.g., a cell, a set of cells, a microbead, a set of microbeads,
`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 microchannel with the first microchannel. The
`fourth microchannelalso 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 memberof the particle
`population between the intersections of the second and the
`third microchannels with the first microchannel.
`
`the computer is
`In this particle sorting embodiment,
`optionally operably linked to thefirst 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 microchannel into the first microchannel to horizon-
`tally or vertically focus the selected memberofthe particle
`population such that the selected member is directed into the
`fourth microchannel
`in response to the detectable signal
`producedby the selected member. Optionally, the instruction
`set further directs simultaneous introduction ofthe at least
`one fluid from the third microchannel by activating a heating
`element (¢.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 microchannel.
`
`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
`elementis typically disposed upstream ofthe 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 includesa fifth microchannel which intersects the
`first microchannel in an intersection region common to the
`second microchannel. In this case, the flow control regulator
`also typically regulates flow of the at least one fluid in the
`second and the fifth microchannels, and the computer
`optionally includes an instruction set for simultaneously
`flowing fluids from the second and the fifth microchannels
`into the first microchannel. Similarly,
`the system also
`optionally includes a sixth microchannel 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 microchannels.
`Furthermore,
`the computer also typically includes an
`instruction set for flowing fluids from the third and the sixth
`microchannels into the first microchannel. Optionally, the
`instruction set directs individual or simultaneous fluid flow
`from the third and sixth microchannels by individually or
`simultaneously activating at least one heating element(e.g.,
`a Joule heating electrode, a conductively coated microchan-
`nel portion,or the like) disposed within each ofthe third and
`sixth microchannels or within at least one well that fluidly
`communicates with each ofthe third and sixth microchan-
`nels.
`
`6
`Manyadditional aspects ofthe 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 (e.g.,
`containers, sealable plastic bags, ete.) and instructions for
`using the devices, ¢.g., to practice the methods herein, are
`also contemplated.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`15
`
`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 showsa 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. 3 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 of U937cells
`that were not treated to induce apoptosis. In this case, the
`bottom line indicates live cell count (Calcein), while the top
`line indicates the presence of apoptotic cells (Annexin-V-
`Cy5). As can be seen, a few apoptotic cells are present
`within the control experiment.
`FIG. 7 is a data graph showing an analysis of U937 cells
`treated with Campthotecin to induce apoptosis. The top trace
`includes a much greater number of peaks representing
`apoptotic cells, and particularly as a percentageoftotal cells
`in the analysis, e.g., as compared to the lower line.
`FIG. 8 is a data graph showing result