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`ABS Global, Inc. and Genus ple — Ex. 1018, p. 1
`
`

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`ABS Global, Inc. and Genusple — Ex. 1018, p. 2
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`INEOca
`Bib Data Sheet
`
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`COMMISSIONER FOR PaTents
`eeee anene
`WW,
`CONFIRMATIONNO.8907
`
`Page 1 ofI
`
`FILING DATE
`
`SERIAL NUMBER GROUPARTUNIT|ATTORNEY05/14/2000
`
`09/569,747
`tai
`DOCKET No.
`RULE
`01-050510US
`
`TINUING DATA *8**tteeannnannnnanamnann
`
`STATE OR|SHEETS TOTAL
`
`COUNTRY|DRAWING|CLAIMS
`CA
`30
`69
`
`APPLICANTS
`H, GARRETT WADA, ATHERTON, CA;
`ANNE R. KOPF-SILL, PORTOLA VALLEY,CA;
`MARUJA LIISA ALAJOKI, PALO ALTO, CA;
`J. WALLACE PARCE, PALO ALTO,CA;
`BENJAMIN N, WANG, SAN FRANCISCO,CA;
`ANDREA W, CHOW,LOS ALTOS,CA;
`ROBERT S. DUBROW, SAN CARLOS, CA;
`
`|
`
`FILING FEE |
`RECEIVED |No.____s to charge/credit DEPOSIT ACCOUNT
`.__ forfollowing:
`
`Cl all Fees
`C) 4.16 Fees(Filing )
`Q 1.17 Fees ( Processing Ext. of
`ime
`
`[ creait
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`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:22)
`ABS Global, Inc. and Genus ple — Ex. 1018, p. 3
`
`

`

`*<H8786i“f”>Hea
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`UTILITY PATENT APPLICATION TRANSMITTALNO FEE
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`STANT COMMISSIONERFOR. .. TENTS
`PATENT APPLICATION
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`SASthington, D.C. 20231
`Se3
`= °ransmitted herewith for filing is the
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`=e
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`divisional application of
`prior application information: Examiner
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`patent application of
`continuation application of
`continuation-in-part patent application of
`
`[X]
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`]]
`
`Inventor(s)/Applicant Identifier: H. Garrett Wadaetal.
`
`For: FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS
`
`[]
`
`[x]
`
`nly: The entire disclosure of the prior application, from which an oath or
`TION or DIVISIO
`For CO
`declaration is supplied is considered a part of the disclosure of the accompanying continuation or divisional application and is
`hereby incorporated by reference. The incorporation can be relied upon whena portion has been inadvertently omitted from
`the submitted application parts.
`This application claims priority from each of the following Application Nos./filing dates: 60/134,472 filed May17, 1999, the
`disclosure(s) of which is (are) incorporated by reference.
`Ld
`]
`Please amend this application by adding the following before the first sentence: “This application is a[ ] continuation [
`=
`continuation-in-part of and claims the benefit of U.S. Application No.
`, the disclosure of which is incorporated
`ei
`by reference for all purposes.”
`Enclosed are:
`(sl
`59 pages ofthe description (includingspecification,
`claims and abstract)
`ta]
`30 sheet(s) of { ] formal [x] informal drawing(s)
`[x]
`0} abstract
`[x]
`53 numberofclaims
`ist
`an assignmentof the inventionto
`[x]
`a[) signed [x] unsigned Declaration
`[-]
`a Power of Attorney
`[J
`a certified copy of a
`[x]
`cover sheet
`[=]
`Information Disclosure Statement under 37 CFR 1.97
`=
`[x]
`
`{]
`(J
`
`[x]
`[x]
`{]
`
`Li
`
`a petition to extend time to respond in the parent
`application
`notification of change of {
`] powerof attorney [
`]
`correspondenceaddressfiled in prior application
`Patent Application Filing Acknowledgementpostcard.
`1___extra copy ofthis sheet is enclosed
`a verified statement to establish small entity status
`under 37 CFR 1.9 and 37 CFR 1.27
`( ) is enclosed
`() was filed in the prior application and
`small entity statusis still proper and desired
`Preliminary amendment
`
`application
`
`Application Data Entry Sheet
`
`In view of the Unsigned Declaration asfiled with this application and pursuant to 37 CFR §1,53(f),
`Applicant requests deferral of the filing fee until submission of the Missing Parts of Application.
`DO NOT CHARGETHE FILING FEE AT THIS TIME.
`
` Christopher C.
`
`Sap
`
`By:
`
`Reg No.: 45,073
`Attorney for Applicant
`
`CORRESPONDENCE ADDRESS:
`Attomey DocketNo._ssOT-050510US
`LAW OFFICES OF JONATHAN ALAN QUINE
`Client Reference No.
`1OO/07410
`P.O. Box 458
`Express Mail” Label No. EL328700805US
`Customer No. 22798
`Alameda, CA 94501
`Date of Deposit: May 11, 2000
`Telephone: (510) 337-7871
`IAA
`Fax: (510) 337-7877
`I hereby certify that this is being deposited with the United States Postal
`Service “Express Mail Post Office to Addressee” service under 37 CFR 1.10
`onthe date indicated above, addressed to: Assistant Commissioner for
`22798
`Patents Washjngton,
`Box Patent
`Application,
`D.C. 20231
`PATENT TRADEKARK OFFICE
`
`Andrew Merit
`
`(out AS
`
`| ls
`
`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:23)
`ABS Global, Inc. and Genus ple — Ex. 1018, p. 4
`
`

`

`
`
`a3
`=a
`|“3
`
`Sia?
`
`eatede
`
`Inventor Information
`
`inventor One Given Name::
`Family Name::
`Postal Address Line One::
`City::
`State or Province::
`Country:
`Postal or Zip Code::
`Citizenship Country::
`
`Inventor Two Given Name::
`Family Name::
`Postal Address Line One::
`City::
`State or Province::
`Country::
`Postalor Zip Code::
`Citizenship Country::
`
`Inventor Three Given Name::
`Family Name::
`Postal Address Line One::
`City::
`State or Province:
`Country:
`Postal or Zip Code::
`Citizenship Country::
`
`Inventor Four Given Name::
`Family Name::
`Postal Address Line One::
`City::
`State or Province::
`Country:
`Postal or Zip Code::
`Citizenship Country:
`
`Inventor Five Given Name::
`Family Name::
`Postal Address Line One::
`City:
`State or Province::
`Country::
`Postal or Zip Code::
`Citizenship Country:
`
`H. Garrett
`Wada
`55 Victoria Drive
`Atherton
`California
`USA
`94027
`the United States
`
`Anne R.
`Kopt-Sill
`30 Minoca Road
`Portola Valley
`California
`USA
`94028
`the United States
`
`Marja Liisa
`Alajoki
`2774 Randers Court
`Palo Alto
`California
`USA
`94306
`Finland
`
`ae
`
`J. Wallace
`Parce
`754 Los Robles Avenue
`Palo Alto
`California
`USA
`94306
`the United States
`
`Benjamin N.
`Wang
`474 Ramsell Street
`San Francisco
`California
`USA
`94132
`the United States
`
`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:24)
`ABS Global, Inc. and Genus ple — Ex. 1018, p. 5
`
`

`

`Inventor Six Given Name::
`Family Name::
`Postal Address Line One::
`City::
`State or Province::
`Country:
`Postal or Zip Code::
`Citizenship Country::
`
`Andrea W.
`Chow
`670 Cuesta Drive
`Los Altos
`California
`USA
`94024
`the United States
`
`Inventor Seven Given Name::_—-Robert S.
`
`Family Name::
`Dubrow
`Postal Address Line One::
`766 Orange Avenue
`City:
`San Carlos
`State or Province::
`California
`Country::
`USA
`Postal or Zip Code::
`94070
`Citizenship Country::
`the United States
`
`CorrespondenceInformation
`
`Correspondence Customer Number::
`E-mail::
`
`22798
`jaquine @ quinelaw.com
`
`Application Information
`
`Title Line One::
`Title Line Two::
`Total Drawing Sheets:
`Formal Drawings?::
`Application Type::
`Docket Number::
`
`FOCUSING OF MICROPARTICLESIN
`MICROFLUIDIC SYSTEMS
`30
`no
`Utility
`01-050510US
`
`Representative Information
`
`Representative Customer Number:
`
`22798
`
`Continuity Information
`
`This application is a::
`> Application One::
`Filing Date::
`
`non-provisionalof
`60/134,472
`May 17, 1999
`
`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:25)
`ABS Global, Inc. and Genus ple — Ex. 1018, p. 6
`
`

`

`Attomey Docket No.: 01-050510/PC
`Client Reference No.: 100/07410
`
`INTERNATIONAL/US PATENT APPLICATION
`
`§
`
`FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS
`
`Inventor(s):
`
`H. Garrett Wada, a citizen of the United States, residing at 55 Victoria
`Drive, Atherton, CA 94027.
`
`Anne R. Kopf-Sill, a citizen of the United States, residing at 30
`Minoca Road, Portola Valley, CA 94028.
`
`Marja Liisa Alajoki, a citizen of Finland, residing at 2774 Randers
`Court, Palo Alto, CA 94303.
`
`J. Wallace Parce, a citizen of the United States, residing at 754 Los
`Robles Avenue, Palo Alto, CA 94306.
`
`Benjamin N. Wang,a citizen of the UnitedStates,residing at 474
`Ramsell Street, San Francisco, CA 94132.
`
`Andrea W. Chow,a citizen of the United States, residing at 670 Cuesta
`Drive, Los Altos, CA 94024.
`
`Robert S. Dubrow,a citizen of the United States, residing at 766
`Orange Avenue, San Carlos, CA 94070.
`
`15
`
`20
`
`25
`
`30
`
`Assignee:
`
`Caliper Technologies Corp., 605 Fairchild Drive, Mountain View, CA
`94043
`wt
`
`As Filed: May 11, 2000
`
`
`
`
`
`
`35
`
`Entity:
`Large
`
`
`
`
`P.O. Box 458
`Alameda, CA 94501
`Internet address: www.quinelaw.com
`
`
`Correspondence Address:
`
`THE LAW OFFICES OF JONATHAN ALAN QUINE
`
`Phone: (510) 337-7871
`Fax: (510) 337-7877
`Email: jaquine@quinelaw.com
`
`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:26)
`ABSGlobal, Inc. and Genusple — Ex. 1018, p. 7
`
`

`

`PATENT
`Attorney Docket No.: 01-050510
`Client Reference No.: 100/07410
`
`iAaSoyan
`
`wotHat
`
`FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS
`
`COPYRIGHT NOTIFICATION
`Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this
`
`disclosure contains material whichis subject to copyright protection. The copyright
`
`ownerhas no objection to the facsimile reproduction by anyoneofthe patent
`
`documentor patentdisclosure, as it appears in the Patent and Trademark Office patent
`
`file or records, but otherwise reserves all copyright rights whatsoever.
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`This applicationis related to and claimspriority to and the benefit of
`
`provisional application 60/134,472,filed 5/17/99, Wada etal., “Focusing of
`Microparticles in Microfluidic 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.
`
`BACKGROUND OF THE INVENTION
`A variety ofcell-based assays are of considerable commercial
`
`relevance in screening for modulators of cell-based activity. For example,
`compounds whichaffect 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, hormonalregulation 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 commonto
`many examples of programmedcell death (or PCD)led to the:suggestion ofa
`common mechanism,and the term “apoptosis” was definedto include both the
`morphological features and the mechanism common to such programmedcell death
`(Kerr et al., Br. J. Cancer 26:239). This concept was extended bythe finding that
`nuclear DNA fragmentation correlates well with apoptotic morphology (Arendsetal.,
`
`10
`
`I
`
`25
`
`30
`
`1
`
`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:27)
`ABSGlobal, Inc. and Genus ple — Ex. 1018, p. 8
`
`

`

`Am. J. Pathol. 136:593 (1990)), and the scientific literature contains many examples
`
`of PCD accompanied bythese features. There are also clear examples of PCD in the
`
`absence of apoptotic morphology or DNA fragmentation (Clarke, Anat. Embryl.
`
`181:195 (1990), Martin et al, J. Cell Biol. 106:829 (1988), and Ishigamietal., J.
`
`Immunol. 148:360 (1992)).
`
`Cell-based assay systems model relevant biological phenomena, and
`
`have generally been widely adopted as screening assays, e.g., when screening for a
`
`compound's effect(s) on apoptosis or other biological phenomena. Pioneering
`
`technology providing cell- and other particle-based microscale assays are set forth in
`
`Parceetal. “High Throughput Screening Assay Systems in Microscale Fluidic
`
`Devices” WO 98/00231; in PCT/US00/04522,filed February 22, 2000, entitled
`
`
`
`“Manipulation of Microparticles In Microfluidic Systems,” by Mehta etal.; and in
`
`PCT/US00/04486,filed February 22, 2000, entitled “Devices and Systems for
`
`Sequencing by Synthesis,” by Mehtaetal.
`Othercell-based assays include various methods for the preparative or
`
`15
`
`analytic sorting of different types of cells. For example,cell panning generally
`involves attaching an appropriate antibodyor othercell-specific reagent to a solid
`support and then exposing the solid support to a heterogeneouscell sample. Cells
`possessing, e.g., the corresponding membrane-bound antigen will bind to the support,
`leaving those lacking the appropriate antigenic determinant to be washed away. Other
`well-known sorting methodsinclude those using fluorescence-activated cell sorters
`(“FACSs”). FACSsfor use in sorting cells and certain subcellular components such
`as molecules of DNAhave 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
`LampIllumination,” 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
`otherparticles 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-throughputorultra high-throughput screening assay systems. For
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`ABSGlobal, Inc. and Genusple — Ex. 1018, p. 9
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`

`

` example,one difficulty in flowing assay systemsis that, during pressure-based flow
`of fluids inchannels, non-uniformflowvelocities areexperienced. “Faster fluidand
`material flow is observed in the center of a moving fluid stream than on the edgeof a
`
`moving fluid stream. This non-uniform flow velocity reduces throughputfor flowing
`
`assays, because assay runs have to be spaced wellapart in the fluid stream to prevent
`overlap of materials movingatdifferentvelocities.
`;
`Accordingly,it would be advantageous to provide mechanismsfor
`facilitating cell-based assays, including cell sorting techniques, especially in
`
`microscale systems. Additional microscale assays directed at subcellular components,
`
`such as nucleic acids would also be desirable. The present invention provides these
`
`and other features which will become clear upon consideration of the following.
`
`SUMMARYOF THE INVENTION
`Thepresent invention relates to methodsoffocusing particles in
`microchannels,e.g., to improve assay throughput,to sort particles, to countparticles,
`
`or the like.
`
`In the methodsofthe 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 noteddifficulties inherent in pressure-based flow of
`particles. Furthermore, the device structuresof the present invention are optionally
`integrated with other microfluidic systems. Other reactions or manipulations
`involvingcells, other particles, or fluids upstream ofthe detection zone are also
`optionally performed, e.g., monitoring druginteractions with'bells or otherparticles.
`In one aspect,the invention provides methods of providing
`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 performingthe flowingstep, the particles are optionally sampled
`with at least one capillary element, e.g., by dipping the capillary elementinto a well
`containing the particles on a microwell plate and drawing the particles into,e.g.,
`reservoirs, microchannels, or other chambers ofthe device. The particles (e.g., a cell,
`
`
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`atonhy—
`feaodhd
`thetHitih
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`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 microchannelto provide substantially
`3
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`ABSGlobal, Inc. and Genus ple — Ex. 1018, p. 10
`
`

`

`uniform flow velocity to the particles in the first microchannel. Particles are
`gpsienally focused usingone¢one or morefluid directiononcomponents (e.g., a fluid
`saTE EETTTeringerrreneee
`‘pressure|reforce modulator, anelectrokinetic forcemodulator, aacapillaryforce
`modulator,afluidwickingelement, orthelike). Additional options include sorting,
`‘detecting «or otherwise manipulating the focusedparticles.
`The particles are horizontally focused in the microchannel, e.g., by
`
`aA
`
`ef)
`
`a?
`
`10
`
`ten
`
`introducing a low density fluid and a high density fluid into the microchannel, causing
`the particles to be 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 flowingparticles. The particles are vertically or
`horizontally focused in the microchannel, e.g., by simultaneously introducing fluid
`flow from two opposing microchannelsinto the first microchannel during flow of the
`particlesin the first channel. Vertical focusing is also optionally achieved to one side
`of a microchannel by simultaneously introducingfluid flow,from, e.g., a second
`microchannelinto the first microchannel during flow of thé*particles iin thefirst
`microchannel.
`
`atMon8
`eBeaeeleaiv
`ony
`aae
`In anotheraspect, the invention also provides particle washing or
`18Lhwa
`exchange techniques. For example, focused cells or other particles are optionally
`20
`washedfree of diffusible material by introducingadiluentinto the first microchannel
`from at least a second channel and removingthe 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
`channelsare also optionally used to further wash theparticles. For example, in one
`aspect the methodsofthe invention include simultaneously introducing the diluent
`into the first microchannel from the second microchanneland a fourth microchannel,
`
`25
`
`where the second and fourth microchannelintersectthe first microchannel at a
`
`30
`
`commonintersection region. Optionally, the methods include sequentially
`introducing the diluentinto the first microchannel from the second microchannel and
`a fourth microchannel, wherein the second and fourth microchannels intersect thefirst
`
`microchannelat an offset intersection region. The diffused productis typically
`4
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`(cid:36)(cid:37)(cid:54)(cid:3)(cid:42)(cid:79)(cid:82)(cid:69)(cid:68)(cid:79)(cid:15)(cid:3)(cid:44)(cid:81)(cid:70)(cid:17)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:42)(cid:72)(cid:81)(cid:88)(cid:86)(cid:3)(cid:83)(cid:79)(cid:70)(cid:3)(cid:177)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:20)(cid:27)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:20)
`ABSGlobal, Inc. and Genus ple — Ex. 1018, p. 11
`
`

`

`removedthrough the third microchannel andafifth microchannel, which third and
`
`fifth microchannelsintersect the first microchannel at a commonintersection region.
`
`In further washing steps, the diluent is introduced through sixth and seventh
`microchannels which intersect the first microchannel at a commonintersection. The
`
`resulting further diluted diffused product is removed through eighth and ninth
`
`microchannels, whichintersectthe first microchannel at a commonintersection.
`
`Diluentis optionally introduced into the first microchannel by pressure or
`electrokinetic flow.
`
`In one preferred assayof the invention, the particles are cells and the
`
`10
`
`methodincludes performing a TUNELassay or an Annexin-V assay onthecells in
`
`
`
`the channel to measure apoptosis.
`Integrated systems for performing the above methods,including the
`particle sorting embodiments, are also provided.
`Anintegrated system for providing substantially uniform flow velocity
`
`15
`
`to flowing members ofat least one particle population in a microfluidic device
`optionally includes a bodystructure that includes atleast a first microchannel
`disposed therein.Afirst fluid direction component(¢.g., a fluid pressure force
`modulator) is typically coupledto the first microchannel for inducing flow ofa fluidic
`material that includes the members ofthe at least one particle populationin thefirst
`microchannel. Thefirst fluid direction component generally induces non-uniform
`
`20
`
`flow. A sourceofat least one fluidic material is optionally fluidly coupledto thefirst
`
`microchannel. The system also optionally includes at least a second microchannel
`that intersects the first microchannelfor introducing at least one fluid into thefirst
`microchannelto horizontally or vertically focus the members oftheat least one
`particle populationin the first microchannel. Theat least onefluid is optionally
`introducedusing a secondfluid direction componentthat 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 ofthe 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 oftheat least one particle population in thefirst
`microchannel and simultaneous introduction ofthe at least one fluid from the second
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`ABSGlobal, Inc. and Genus ple — Ex. 1018, p. 12
`
`

`

`
`
`
`microchannelinto the first microchannelis optionally also operably coupled to a fluid
`movementsystem for directing flow of materials in the microchannels.
`As a further option,this integrated system additionally includesat least
`a third microchannel which intersects the first microchannel in an intersection region
`
`commonto the second microchannel. The flow control regulatorof this system
`optionally further regulates flow ofthe at least onefluid in the second andthethird
`microchannels.
`In this embodiment, the computertypically also includes an
`instruction set for simultaneously flowing fluids from the secondandthird
`microchannels into thefirst microchannel.
`In particle washing systems,typically, at least fourth and fifth channels
`whichintersect the first microchannel at a commonintersection downstream of the
`second and third microchannels are provided. The computer further includes an
`instruction set for simultaneously flowing material from the first microchannelinto
`the fourth andfifth microchannels. Sixth and seventh microchannels whichintersect
`the first microchannel at a commonintersection downstream ofthe fourth andfifth
`microchannels, with the computer further comprising an instruction set for
`simultaneously flowing material from the sixth and seventh microchannelsinto the
`first microchannelare optionally provided. Similarly, eighth and ninth microchannels
`whichintersect the first microchannel at a commonintersection downstream of the
`sixth and seventh microchannels, the computer further including an instruction set for
`simultaneously flowing material from thefirst microchannelinto the eighth and ninth
`microchannels are optionally provided.
`Theintegrated system optionally includes sources for any reagent or
`particle used in the methodsnoted 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 moresources 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 controlcell, gne or more sources of a test cell, ete.
`Signal detector(s)mounted proximalto the first microchannel for
`detecting a detectable signal produced by one or more ofthe members ofthe atleast
`oneparticle population in the microchannelare typically providedin the integrated
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`ABSGlobal, Inc. and Genus ple — Ex. 1018, p. 13
`
`

`

`systems of the invention. The detector also optionally includes,e.g., a fluorescent
`
`excitation source and a fluorescent emission detection element. Optionally, the
`
`computer is operably linked to the signal detector and hasan instruction set for
`
`converting detected signal information into digital data.
`
`The integrated system of the present invention is also 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 microbeads, a
`
`molecule, a set of molecules, or the like).
`
`In this embodiment,the integrated system
`
`typically additionally includes a third and a fourth microchannel whichintersect the
`first microchannel downstream from the intersection of the second microchannel with
`
`the first microchannel. The fourth microchannel also generally intersectsthefirst
`
`microchannel downstream from the intersection ofthe third microchannel with the
`
`first microchannel. The flow control regulator of this system optionally further
`
`regulates flow ofthe at least onefluid in the third or the fourth microchannels.
`Furthermore,the signal detectortypically detects a detectable signal produced by a
`
`selected memberof the particle population between the intersections of the second
`and the third microchannels with the first microchannel.
`
`operablylinkedto the firstorotherfluiddirectioncompoadtthy, theflowcontrol
`
`In this particle sorting embodiment, the computer is optionally
`
`regulator, and the signal detector. Additionally, the instruction set typically directs
`simultaneous introduction ofthe at least one fluid from the third microchannelinto
`
`the first microchannelto horizontally orvertically focus the selected memberofthe
`particle population suchthat the selected memberis directed into the fourth
`microchannelin response to the detectable signal producedbythe selected member.
`Optionally, the instruction set further directs simultaneous introduction ofthe 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 microchannelor a well that fluidly communicates with the third
`microchannel.
`
`In another embodiment,at least a portion of the first microchannel
`optionally includes a separation element disposedtherein. The separation element
`optionally includes, e.g., two sides andat least a portion of the separation elementis
`7
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`ABSGlobal, Inc. and Genusple — Ex. 1018, p. 14
`
`

`

`
`
`typically disposed upstream ofthe fourth microchannel. In this embodiment, a
`
`selected memberofthe particle population is generally directed to one of the two
`
`sides of the separation elementand into the fourth microchannelthatintersects the
`first microchannel in responseto the detectable signal produced by the selected
`member.
`
`The integrated system for use in particle sorting also optionally
`includes a fifth microchannel whichintersects the first mi