US008529161B2
`
`a2) United States Patent
`US 8,529,161 B2
`(0) Patent No.:
`Sep. 10, 2013
`(45) Date of Patent:
`Gilbertet al.
`
`(54) MULTILAYER HYDRODYNAMIC SHEATH
`FLOW STRUCTURE
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`(75)
`
`Inventors: John R. Gilbert, Brookline, MA (US);
`Manish Deshpande, Canton, MA (US);
`Bernard Bunner, Watertown, MA (US)
`
`(73)
`
`Assignee: Cytonome/ST, LLC, Boston, MA (US)
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`US.C. 154(b) by 201 days.
`
`(21) Appl. No.: 13/179,084
`
`(22)
`
`Filed:
`
`Jul. 8, 2011
`
`(65)
`
`Prior Publication Data
`
`US 2012/0009025 Al
`
`Jan. 12, 2012
`
`3/1972 Randolph ww. 250/364
`3,649,829 A *
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`7/1988 Gohde etal. .
`.. 209/3.1
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`.
`we 356/73
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`9/1990 Zold ........
`.. 250/461.1
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`1/1991 Ohkietal.
`oe. 356/246
`4,983,038 A *
`
`.
`7/1991 North, Jr.
`we 356/73
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`we 356/72
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`..
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`6,365,106 BL*
`
`(Continued)
`FOREIGN PATENT DOCUMENTS
`0070080 Al
`11/2000
`03078972 Al
`9/2003
`
`WO
`WO
`
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`(1)
`
`(52)
`
`Continuation of application No. 12/610,753, filed on
`Nov. 2, 2009. now Pat. No. 7,997,831, which is a
`continuation of application No. 11/998,557, filed on
`Nov. 30, 2007, now Pat. No. 7,611,309, which is a
`continuation of application No. 10/979,848, filed on
`Nov.1, 2004, now Pat. No. 7,311,476.
`
`Provisional application No. 60/516,033, filed on Oct.
`30, 2003.
`
`Int. Cl.
`B65G 51/00
`US. Cl.
`USPC wee 406/198; 406/86; 406/94; 406/195;
`356/246; 435/174
`
`(2006.01)
`
`(58) Field of Classification Search
`USPC wee 406/86, 93, 94, 195, 198; 356/246;
`435/174
`See application file for complete search history.
`
`Primary Examiner — Joseph A Dillon,Jr.
`(74) Attorney, Agent, or Firm — McCarter & English, LLP;
`David R. Burns
`
`ABSTRACT
`(57)
`A microfabricated sheath flow structure for producing a
`sheath flow includes a primary sheath flow channel for con-
`veying a sheath fluid, a sample inletfor injecting a sample into
`the sheath fluid in the primary sheath flow channel, a primary
`focusing region for focusing the sample within the sheath
`fluid anda secondary focusing region for providing additional
`focusing of the sample within the sheath fluid. The secondary
`focusing region may be formedbya flow channelintersecting
`the primary sheath flow channel to inject additional sheath
`fluid into the primary sheath flow channel from a selected
`direction. A sheath flow system may comprise a plurality of
`sheath flow structures operating in parallel on a microfluidic
`chip.
`
`20 Claims, 12 Drawing Sheets
`
`
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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:19)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)
`ABS Global, Inc. and Genusple — Ex. 1001, p. 1
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`US 8,529,161 B2
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`Page 2
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`(56)
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`References Cited
`
`U.S. PATENT DOCUMENTS
`.
`Oeeo BI oot puny “a sestvnssertenen 356/246
`6's37501 B1® 3/2003 Holletal..
`493/537
`6576194 BL®
`6/2003 Holldal 499/81
`6592821 BL
`7/2003 Wadaetal.
`....escse 356/246
`6,674,525 B2*
`1/2004 Bardell et al.
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`3/2004 Mavliev .........
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`6,710,874 B2*
`7,116,407 B2* 10/2006 Hansen etal.
`356/73
`1/2007 Bohmetal. wc 435/325
`7,157,274 B2*
`7,223,371 B2
`5/2007 Hayengaetal.
`7,311,476 B2
`12/2007 Gilbert etal.
`7,355,696 B2*
`4/2008 Muethetal. o......c 356/244
`
`......ccccee 494/36
`7,402,131 B2*
`7/2008 Muethet al.
`7,442,339 B2* 10/2008 Sundararajan et al.
`.... 422/82.05
`
`7,452,726 B2* 11/2008 Chouetal. oo. 436/63
`7,553,453 B2*
`6/2009 Guetal. icc 422/537
`7,611,309 B2
`11/2009 Gilbert etal.
`......... 436/63
`7,638,339 B2* 12/2009 Sundararajan etal.
`
`........ 422/73
`7,641,856 B2*
`1/2010 Padmanabhan etal.
`7,751,040 B2*
`7/2010 Chang et al. 1... 356/246
`7,760,351 B2*
`7/2010 Cox etal. cece 356/246
`8/2010 RiCh weccssscseessssesssneees 422/81
`7,776,268 B2*
`
`7,802,686 B2™ 9/2010 Takagi et al...
`209/172.5
`
`7,833,421 B2™ 11/2010 Huymann .....
`. 210/748.01
`12/2010 Sundararajan
`«1.0.0... 422/50
`7,850,907 B2*
`8/2011 Tabata et al... 436/174
`7,993,934 B2*
`7,997,831 B2
`8/2011 Gilbert et al.
`8,263,387 B2*
`9/2012 Pagano etal. oe 435/283.1
`8,383,043 B2*
`2/2013 Padmanabhan et al.
`..... 422/68.1
`
`* cited by examiner
`
`(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:19)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)
`ABS Global, Inc. and Genusple — Ex. 1001, p. 2
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`U.S. Patent
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`Sep. 10, 2013
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`US8,529,161 B2
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 3
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`U.S. Patent
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 4
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 5
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 7
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 8
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 9
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`U.S. Patent
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 10
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`ABS Global, Inc. and Genusple — Ex. 1001, p. 14
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`US 8,529,161 B2
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`1
`MULTILAYER HYDRODYNAMIC SHEATH
`FLOW STRUCTURE
`
`RELATED APPLICATIONS
`
`The present invention is a continuation application of U.S.
`patent application Ser. No. 12/610,753, filed Nov. 2, 2009 and
`is a continuation application of U.S. patent application Ser.
`No. 11/998,557, filed Nov. 30, 2007, which, in turn, is a
`continuation application of U.S. patent application Ser. No.
`10/979,848, now U.S. Pat. No. 7,311,476, entitled, “Multi-
`layer Hydrodynamic Sheath Flow Structure” filed Nov. 1,
`2004, which claimspriority to U.S. Provisional Application
`Ser. No. 60/516,033, filed Oct. 30, 2003, the entire content of
`each application is herein incorporated by reference in their
`entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates to a system and method for
`producing a sheath flow in a flow channel. Moreparticularly,
`the present invention relates to a system and method for
`producing a sheath flow in a microchannelin a microfluidic
`device.
`
`BACKGROUNDOF THE INVENTION
`
`10
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`2
`sheath flow channel and a sample inlet for introducing a
`sample to the structure. A sample is introducedto the sheath
`fluid in the primary sheath flow channel via the sample inlet
`and suspended therein. The primary sheath flow channel may
`branch at a location upstream ofa sample inlet to create a flow
`in an upper sheath channel. The primary sheath flow channel
`formsa primary focusing region for accelerating sheath fluid
`in the vicinity of a sample channel connected to the sample
`inlet. The sample channel provides the injected sample to the
`accelerating region, such that the particles are confined in the
`sheath fluid. The primary focusing region further focuses the
`sheath fluid around the sample. The sheath flow then flows to
`a secondary sheath region downstream ofthe primary accel-
`erating region connects the upper sheath channelto the pri-
`mary sheath flow channel to further focus the sample in the
`sheath fluid. The resulting sheath flow forms a focused core of
`sample within a channel.
`The sheath flow structure may be parallelized to provide a
`plurality of sheath flow structures operating in parallel in a
`single system. The parallelized system may have a single
`sample inlet that branches into a plurality of sample channels
`to inject sample into each primary sheath flow channelof the
`system. The sample inlet may be provided upstream ofthe
`sheath inlet. Alternatively, the parallelized system may have
`multiple sample inlets. The parallelized sheath flow structure
`may have a single sheath fluid inlet for providing sheath fluid
`to all of the primary sheath flow channels and/or secondary
`sheath channels, or multiple sheath fluid inlets for separately
`Sheath flow is a particular type of laminar flow in which
`providing sheath fluid to the primary sheath flow channels
`onelayer offluid,or a particle, is surrounded by another layer
`and or secondary sheath channels.
`of fluid on more than one side. The process of confining a
`Accordingtoafirst aspect of the invention, a sheath flow
`particle stream in a fluid is referred to as a ‘sheath flow’
`structure for suspending a particle in a sheath fluid is pro-
`configuration. For example, in sheath flow, a sheath fluid may
`vided. The sheath flow structure comprises a primary sheath
`envelop and pinch a sample fluid containing a number of
`flow channel for conveying a sheath fluid, a sample inlet for
`particles. The flow of the sheath fluid containing particles
`injecting a particle into the sheath fluid conveyed through the
`suspended therein may be narrowed almostto the outer diam-
`primary sheath flow channel, a primary focusing region for
`eter of particles in the center ofthe sheath fluid. Theresulting
`sheath flow flows in a laminar state within anorifice or chan-
`focusing the sheath fluid aroundtheparticle in atleasta first
`direction and a secondary focusing region provided down-
`nel so that the particles are lined and accurately pass through
`stream of the primary focusing region. The secondary focus-
`the orifice or channel in a single file row.
`ing region focuses the sheath fluid aroundtheparticle in at
`Sheath flow is used in many applications whereit is pref-
`least a second direction different from the first direction.
`erable to protect particles or fluids by a layer of sheath fluid,
`for example in applications wherein it is necessary to protect
`particles from air. For example,particle sorting systems, flow
`cytometers and other systems for analyzing a sample, par-
`ticles to be sorted or analyzedare usually supplied to a mea-
`surement position in a central fluid current, which is sur-
`rounded by a particle free liquid sheath.
`Sheath flow is useful because it can position particles with
`respect to sensors or other components and preventparticles
`in the center fluid, which is surrounded by the sheath fluid,
`from touching the sides of the flow channel and thereby
`prevents clogging of the channel. Sheath flow allows for
`faster flow velocities and higher throughput of sample mate-
`rial. Faster flow velocity is possible without shredding cells in
`the centerfluid because the sheath fluid protects the cells from
`shear forcesat the walls of the flow channel.
`Conventional devices that have been employed to imple-
`ment sheath flow have relatively complex designs and are
`relatively difficult to fabricate.
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`SUMMARYOF THE INVENTION
`
`The present invention provides a microfabricated sheath
`flow structure for producing a sheath flow fora particle sort-
`ing system or other microfluidic system. The sheath flow
`structure may comprise a two-layer construction including a
`sheath inlet for introducing a sheath fluid into a primary
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`Accordingto anotheraspect ofthe invention, a sheath flow
`structure for suspending a particle in a sheath fluid comprises
`a first substrate layer including a primary sheath flow channel
`for conveying a sheath fluid and a second substrate layer
`stacked onthefirst substrate layer. The second substrate layer
`includesa first sheath inlet for introducing a sheathfluid to the
`primary sheath flow channel, a sample inlet downstream of
`the first sheath inlet for providing the particle to the primary
`sheath flow channel in a primary focusing region to form a
`sheath flow including the particle surrounded by the sheath
`fluid on at least one side. A first secondary sheath channelis
`formedin thefirst or second substrate layer in communication
`with the primary sheath flow channel. The first secondary
`sheath channel diverts a portion of said sheath fluid from the
`primary sheath flow channel.
`Accordingto still another aspect of the invention, a focus-
`ing region for focusing a particle suspended in a sheath fluid
`ina channelofa sheath flow device is provided. The focusing
`region comprises a primary flow channel for conveying a
`particle suspendedin a sheath fluid anda first secondary flow
`channel
`intersecting the primary flow path for injecting
`sheath fluid into the primary flow channel from above the
`particle to focus the particle away from a top wall of the
`primary flow channel.
`According to another aspect of the invention, a method of
`surrounding a particle on at least two sides by a sheath fluid,
`(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:19)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:24)
`ABS Global, Inc. and Genus ple — Ex. 1001, p. 15
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`US 8,529,161 B2
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`4
`FIG. 11 is a histogram superimposing the fluorescence
`measurements from all eight primary sheath flow channels in
`the system of FIGS. 8A-8B.
`FIG. 12 illustrates the distribution of core sizes for the
`
`sheath flows producedin the primary sheath flow channels in
`the system of FIGS. 8A-8B.
`
`DETAILED DESCRIPTION OF THE INVENTION
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`The present invention provides a system and method for
`producing a sheath flow in a flow channel, such as a micro-
`channel. The present invention will be described belowrela-
`tive to illustrative embodiments. Those skilled in the art will
`appreciate that the present invention may be implemented in
`a numberofdifferent applications and embodiments andis
`not specifically limited in its application to the particular
`embodiments depicted herein.
`As used herein, the term “microfluidic” refers to a system
`or device for handling, processing, ejecting and/or analyzing
`a fluid sample includingat least one channel having micros-
`cale dimensions.
`The terms “channel” and “flow channel” as used herein
`refers to a pathway formedin or through a medium that allows
`for movementoffluids, such as liquids and gases. A “micro-
`channel”refers to a channelin the microfluidic system pref-
`erably have cross-sectional dimensionsin the range between
`about 1.0 um and about 500 um, preferably between about 25
`um and about 250 um and most preferably between about 50
`uum and about 150 um. Oneof ordinary skill in the art will be
`able to determine an appropriate volume and length of the
`flow channel. The ranges are intended to include the above-
`recited values as upperor lower limits. The flow channel can
`have any selected shape or arrangement, examples of which
`include a linear or non-linear configuration and a U-shaped
`configuration.
`FIG.1 illustrates a microfabricated sheath flow structure
`
`3
`comprises the steps of injecting a sheath fluid into a primary
`sheath flow channel diverting a portionofthe sheath fluid into
`a branching sheath channel, injecting the particle into the
`primary sheath flow channel to suspend the particle in the
`sheath fluid to form a sheath flow and injecting the diverted
`portion of the sheath fluid into the sheath flow to focus the
`particle within the sheath fluid.
`According to another aspect of the invention, a method of
`surrounding a particle on at least two sides by a sheathfluid,
`comprises the steps of conveying a sheath fluid through a
`primary sheath flow channel, injecting a particle into the
`sheath fluid conveyed through the primary sheath flow chan-
`nel, focusing the sheath fluid aroundtheparticle in at least a
`first direction and focusing the sheath fluid aroundthe particle
`in at least a seconddirection different from thefirst direction.
`
`According to still another aspect, a sheath flow system is
`provided which comprises a plurality of a sheath flow struc-
`tures operating in parallel on a substrate. Each sheath flow
`structure comprises a primary sheath flow channel for con-
`veying a sheath fluid, a sample channel for injectinga particle
`into the sheath fluid conveyed through the primary sheath
`flow channel, a primary focusing region for focusing the
`sheath fluid aroundtheparticle in at least a first direction and
`a secondary focusing region provided downstream of the
`primary focusing region for focusing the sheath fluid around
`the particle in at least a second direction different from the
`first direction.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 illustrates a sheath flow structure according to an
`illustrative embodimentof the invention.
`FIGS. 2A-2B illustrate a multilayer sheath flow structure
`according to an illustrative embodimentofthe invention.
`FIG. 2C illustrates is a cross sectional view through the
`centerline of the sheath flow structure of FIG. 2A, showing
`the path of an injected particle throughthe structure.
`FIG.3 illustrates the path of a particle through the multi-
`layer sheath flow structure of FIGS. 2A-2C.
`FIG. 4A illustrates the flow profile within the primary
`focusing region and the secondary focusing region during
`operation of the sheath flow structure of FIGS. 2A-2C.
`FIGS. 4B-4Dare detailed cross-sectional views ofthe flow
`
`profiles within the primary sheath flow channelat different
`stages during operation of the sheath flow structure of FIGS.
`2A-2C,
`
`FIGS. 5A-5Cillustrates a multilayer sheath flow structure
`according to an alternate embodimentofthe invention, where
`a sample is injected directly into a focusing region.
`FIG. 6 is a perspective view of a sheath flow structure
`according to another embodimentof the invention.
`FIGS. 7A-7Billustrate a sheath flow structure including a
`sample inlet provided upstream of a sheath flow inlet accord-
`ing to another embodimentof the invention.
`FIGS. 8A-8B illustrate a parallelized sheath flow system
`for producing sheath flow in multiple parallel channels
`according to another embodimentof the invention.
`FIG. 9A is a fluorescent microscope image of a primary
`sheath flow channel downstream from the secondary focusing
`region in the parallelized sheath flow system of FIGS. 8A and
`8B.
`
`FIG.9Bis fluorescent microscope image taken of a sample
`in the primary sheath flow channel of FIG. 9A after focusing
`of the sample.
`FIG. 10 is a histogram diagramming the measured amount
`of fluorescence in the channel observed in FIG. 9B across
`axis-A-A-.
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`10 according to an illustrative embodimentof the invention.
`The sheath flow structure 10 may be used to suspendparticles
`in a sheath fluid flow stream for use in a particle sorting
`system or other microfluidic system. The sheath flow struc-
`ture 10 includes a primary sheath flow channel 12 for con-
`veying sheath fluid through the sheath flow structure 10. A
`flow maybe induced through the primary sheath flow channel
`12 through any means knownintheart, including one or more
`pumps. The sheath flow structure 10 further includes a sample
`inlet 15 for introducing a sample, such as one or more par-
`ticles, to the sheath fluid flowing through the primary sheath
`flow channel 12, so that the sample is surrounded by the
`flowing sheath fluid. The sample inlet 15 may comprise a
`channel, reservoir or other suitable component in communi-
`cation with the primary sheath flow channel 12.
`According to one embodiment, the microfabricated sheath
`flow structure is formed on a microfluidic chip andthe pri-
`mary sheath flow channel and other flow channels formed
`therein are microchannels having microscale dimensions.
`However, one skilled in the art will recognize that the sheath
`flow structure mayalternatively have larger dimensions and
`be formed using flow channels having cross-sectional dimen-
`sions greater than 500 jun. The illustrative sheath flow struc-
`ture can be fabricated in glass, plastics, metals or any other
`suitable material using microfabrication, injection molding/
`stamping, machiningor other suitable fabrication technique.
`After introduction of the sample into the sheath fluid, a
`primary focusing region 17 accelerates and focuses the sheath
`fluid around the injected sample. Preferably, the primary
`focusing region 17 focuses the sheath fluid away from the
`sides and bottom of the sample. A secondary focusing region
`(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:19)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:25)
`ABS Global, Inc. and Genusple — Ex. 1001, p. 16
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`US 8,529,161 B2
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`the sample particles are suspendedin the sheath fluid. Alter-
`19, disposed downstream of the primary focusing region 17
`natively, each of the subchannels 12a, 125 may have a sepa-
`along the primary sheath flow channel, provides additional
`rate inlet, and the separated subchannels may convergein the
`focusing of the sheath fluid around the sample afterthe pri-
`primary focusing region 17.
`mary focusing region performs the primary focusing. Prefer-
`In the primary focusing region 17, the sample particles
`ably, the secondary focusing region 19 focuses the sample in
`injected into the sheath flow are focused away from the sides
`a vertical direction from above the sample.
`and bottom by the sheath flow. As shown, the outlet of the
`Accordingto an illustrative embodiment, the combination
`sample flow channel 16 is in substantially the middle of the
`ofthe primary focusing region 17 and the secondary focusing
`primary focusing region 17, between the outlets of the sub-
`region 19 provides three-dimensional focusing of the sheath
`channels 12a, 124, such that the particles are surrounded by
`fluid around the sample. The resulting sheath flow is sample-
`sheath fluid flowing from the subchannels on both sides ofthe
`focused hydrodynamically on all sides of the sample away
`injected particles and centralized within the sheath fluid flow.
`from the walls ofthe primary sheath flow channel12, with the
`The sheath flow channel 12 in the primary focusing region
`sample being suspendedas a focused core in the approximate
`center of the channel.
`thentapers fromarelatively wide width W atthe outlets ofthe
`15
`subchannels 12a, 124 to a smaller width W'to force the sheath
`The secondary focusing region 19 passes the resulting
`sheath flow in the primary sheath flow channel 12to a particle
`fluid around the suspended sample particles.
`sorting system or other microfluidic system or component in
`After suspension of the sample particles, the sheath flow
`fluid communication with an outlet 19a of the secondary
`then flows from the primary focusing region 17 through the
`focusing region 19. The microfluidic system for receiving the
`sheath flow channel 12, which forms the secondary focusing
`sheath flow may be formed on the samechip or substrate as
`region 19 downstream of the primary focusing region 17.
`the sheath flow structure or a different substrate in fluid com-
`According to an illustrative embodiment,
`the secondary
`munication with the sheath flow structure 10.
`focusing region 19 utilizes sheath fluid to provide secondary
`According to one embodiment, the sheath flow structure
`focusing ofthe sheath flow in a vertical direction after the
`may be formed using a plurality of stacked layers. For
`initial focusing provided by the primary focusing region 17.
`example, FIGS. 2A-2C illustrate a two-layer sheath flow
`For example, as shown in FIGS. 2A-2C,the secondary focus-
`structure 100 for producing sheath flow according to one
`ing region 19 may be formed by secondary sheath channels
`embodimentof the invention. In FIGS. 1 and 2A-2C, similar
`13a, 134 that intersect the primary sheath flow channel 12 in
`parts are indicated by equivalent reference numbers. The
`the secondary focusing region 19. The secondary sheath
`illustrated sheath flow structure 100 has a two-layer construc-
`channels 13a, 136 flow and inject sheath fluid into the primary
`tion including a bottom substrate layer 105 and atop substrate
`sheath flow channel 12 to focus the sample within the sheath
`fluid.
`layer 10a stacked on the bottom substrate layer 105. Those of
`As shown,the inlets to the secondary sheath channels 13a,
`ordinary skill will recognize that any suitable number of
`138, respectively, may intersect the primary sheath flow chan-
`layers can be used. The top substrate layer 10a may have
`nel 12 in an intermediate upstream region between the sheath
`formedtherein a sheath inlet 11 for introducing a sheath fluid
`inlet 11 and the outlet ofthe sample channel 16. Branch points
`to the primary sheath flow channel 12 and a sample inlet 15
`24a, 246 connect each of the secondary sheath channels 13a,
`for introducing a sample to the sheath flow structure. The
`136 to the primary channel12 to divert a portionofthe sheath
`primary sheath flow channel 12 for conveying the sheath fluid
`fluid from the primary sheath flow channel to each of the
`throughthe structure is formed in the bottom layer 106 of the
`secondary sheath channels 13a, 135,
`respectively. The
`two-layer sheath flow structure 100. As shown, the sample
`diverted sheath flow then flows to the secondary focusing
`inlet 15 connects to a sample channel 16, which intersects the
`region 19, wherethe outlets of the secondary sheath channels
`primary sheath flow channel 12 downstream of the sheath
`13a, 136 intersect the primary sheath flow channel 12. Pref-
`inlet 11 to inject a sample, such as a stream ofparticles, into
`erably, the outlets of both secondary sheath channels extend
`a sheath fluid flowing in the primary sheath flow channel 12.
`above and substantially parallel to the fluid flow in the pri-
`While the illustrative two-layer sheath flow structure 100
`mary sheath flow channel 12 in the vicinity of the secondary
`injects the sheath flow and sample particles from a top surface
`focusing region 19. In this manner, secondary sheath fluid
`of the structure, one skilled in the art will recognize that the
`from the secondary sheath channels 13a, 134 enters the pri-
`sheath inlet 11 and sample inlet 15 can be provided in any
`mary sheath flow channel 12 from the sameside as the
`suitable location and have anysuitable size and configuration.
`sample, compressing the suspended sample away from the
`The primary focusing region 17 in the two-layer sheath
`flow structure 100 of FIGS. 2A-2C maybe formedbytaper-
`upper wall of the channel 12 (1.e., in the otherdirection from
`
`ing the primary sheath flow channel 12 fromarelatively wide the main sheath of fluid aroundthe particle).
`width W to a smaller width W' downstream ofthe intersection
`In the illustrative embodiment, branch points 24a, 245
`ext