United States Patent
`Weigl et al.
`
`{15
`
`[11]
`
`[45]
`
`Patent Number:
`
`Date of Patent:
`
`6,159,739
`Dec. 12, 2000
`
`US006159739A
`
`[54] DEVICE AND METHODFOR3-
`DIMENSIONAL ALIGNMENT OF
`PARTICLES IN MICROFABRICATED FLOW
`CHANNELS
`
`[75]
`
`Inventors: Bernhard Weigl; Paul Yager, both of
`Seattle, Wash.; James P. Brody,
`Pasadena, Calif.
`.
`an
`.
`[73] Assignee: University of Washington,Seattle,
`Wash.
`
`OTHER PUBLICATIONS
`. Ce .
`.
`Chmelik, Josef, “Isoelectric focusing field—flow fraction-
`ation” Journal of Chromatography 545, No. 2 (1991).
`Eisert, W.G. et al. (1975), “Simple flow microphotometerfor
`rapid cell population analysis,” Rev. Sci. Instrum. 46(8):
`1021-1024.
`Sobek et al. (1993), “A Microfabricated Flow Chamberfor
`Optical Measurements in Fluids,” in Proc. of the IEEE
`Micro Electro Mechanical Systems Workshop, Fort Lauder-
`dale, Florida, Feb. 1993, pp. 219-224.
`
`[21] Appl. No.: 08/823,747
`
`(List continued on next page.)
`
`
`
`Filed:
`Mar. 26, 1997
`Primary Examiner—Maureen M. Wallenhorst
`[22]
`Tint, C0? eecceccccccsssssssensccsscssessenuneeeeeeescen GOLN 35/08 Berns Agent, or Firm—Greenlee, Winner and Sullivan,
`PSD]
`
`sever 436/52; 436/53; 436/165;
`[52] U.S. Cl. .......
`436/172; 422/81; 422/82; 422/82.05; 422/82.08;
`[57]
`ABSTRACT
`356/246
`The present invention provides a sheath ow module made
`
`[58] fromafirst plate of material having formed therein a laminarField of Search wo.eee 436/52, 53, 63,
`436/164, 165, 172; 422/81, 82, 82.05, 82.08,
`fluid flow channel; at least two inlets, eachinlet joining the
`82.09; 356/246
`laminar flow channel at a junction, the first inlet junction
`being wider than the second inlet junction, and an outlet
`from the flow channel. A second plate, e.g. a transparent
`cover plate, seals the module and allows for optical mea-
`surements. A first inlet allows for introduction of a first fluid
`U.S. PATENT DOCUMENTS
`into the flow channel. Thefirst fluid is the sheath fluid. A
`.
`secondinlet allows for introduction of a secondfluid into the
`:
`toseaot ies Cuedmanetal mene0 sheathfluid whileit is flowing through the flow channel.The
`
`....scsccssssssesesesseneesee 209/12
`4,894,146
`1/1990 Giddings
`second fluid is the center fluid. Because the second inlet
`
`204/299 R
`3/1990 Pace wc.
`4,908,112
`junction is narrower than thefirst inlet junction, the center
`
`
`venee 356/246
`4,983,038
`1/1991 Ohki etal.
`fluid becomes surroundedon both sides by the sheath fluid.
`
`4/1991 Oki et ab. ce
`eceseeseeeeeeeeeeeeees 356/73
`5,007,732
`After all fluids have been introduced and sheath flow has
`
`-. 210/695
`5,039,426
`8/1991 Giddings......
`been achieved,
`the depth of the flow channel can be
`73/864.85
`5,079,959
`1/1992 Miyakeetal.
`decreased,
`leading to vertical hydrodynamic focusing.
`
`10/1992 Kosaka ......ssssesnrsesnnennees 356/243
`5,159,403
`Optionally, the width of the flow channel can be decreased,
`leading to horizontal hydrodynamic focusing. The decrease
`in depth and width can be gradual or abrupt. The device of
`the present invention can function in two modes, the sheath
`flow modeandthe particle injector mode, depending on the
`relative densities of the sheath fluid, the center fluid, and any
`get ts
`:
`particles in eitherfluid.
`
`_
`[56]
`
`.
`References Cited
`
`(List continued on next page.)
`
`FOREIGN PATENT DOCUMENTS
`ae
`,
`,
`0 288 029 A2
`European Pat. Off.
`.
`4/1988
`0 294701
`European Pat. Off..
`9/1992
`25 21 236 Al
`11/1976 Germany.
`WO 97/00442
`1/1997 WIPO .
`
`48 Claims, 12 Drawing Sheets
`
`La 3c.
`
`lH
`
`1 <
`
`-3C33
`
`20
`
`DD
`
`5
`
`ll
`
`7
`
`al
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 1
`
`

`

`6,159,739
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`8/1993 Caldwell et al. voces 210/748
`5,240,618
`. 435/287.2
`§,674,743
`......
`we. 436/172
`2/1998 Yageret al.
`5,716,852
`...
`.. 356/246
`3/1998 Altendorf et al.
`5,726,751
`
`5/1998 van den Enghet al.
`.
`.. 436/172
`5,747,349
`8/1999 Yager et al.
`.........
`... 210/634
`5,932,100
`9/1999 Weigl et al.
`..
`. 436/52
`5,948,684
`5,972,710 10/1999 Weigl et al. occ ceeeceeee 436/34
`OTHER PUBLICATIONS
`
`10/1997 Ulmer...........
`
`
`Gravesen, P. et al. (1993), “Microfluidics —a review,” J.
`Micromech. Microeng 3:168-182.
`Kikuchi, Y.et al. (1992), “Optically Accessible Microchan-
`nels Formed in a Single—Crystal Silicon Substrate for Stud-
`ies of Blood Rheology,” Microvascular Res. 44:226-240.
`
`Miyake, R. et al. (1991), “A Development of Micro Sheath
`Flow Chamber,” in Proceedings of the IEEE Micro Electro
`Mechanical Systems Workshop, Nara, Japan, Jan. 1991, pp.
`265-270.
`
`Verpoorte, E. et al. (1993), “A silicon flow cell for optical
`detection in miniaturized total chemical analysis systems,”
`Sensors and Actuators B 6:66—70.
`
`Wilding, P. et al. (1994), “Manipulation and Flow of Bio-
`logical Fluids in Straight Channels Micromachined in Sili-
`con,” Clin. Chem. 40(1):43-4+7.
`
`Sobek,D. et al. (1994), “Microfabricated Fused Silica Flow
`Chambers for Flow Cytometry,” Solid State Sensor and
`Actuators Workshop, Hilton Head, South Carolina, Jun.
`1994, 4 pp.
`
`(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:24)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)
`ABS Global, Inc. and Genusplc — Ex. 1005, p. 2
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 1 of 12
`
`6,159,739
`
`PRIOR ART
`
`44
`
`435
`
`40
`
`2
`
`4I
`
`56
`42
`
`4|
`
`Figure |
`
`Figure 2
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 3
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 2 of 12
`
`6,159,739
`
`6
`
`3A
`
`(1 H+X
`
`9g SI
`
`1 L
`
`|
`
`SC
`
`Nl
`
`7
`
`2I
`
`8
`
`Figure 3A
`
`i
`
`2|
`
`3
`
`Figure 3B
`
`54
`
`8
`
`Figure 3C
`
`53
`
`|
`
`5
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 4
`
`l
`
`5
`
`3A
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 3 of 12
`
`6,159,739
`
`
`
`Figure 4B
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 5
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 4 of 12
`
`6,159,739
`
`mM
`
`Q
`Ww
`
`oO
`m®
`
`a
`
`Q
`Te)
`
`|
`
`|
`
`<
`
`om
`
`Sb
`
`
`
`
`
`SS Se -——-Y«-O LO
`
`2
`
`3
`fs om
`Ti
`
`WV
`
`—
`y
`
`lib
`
`5
`
`mo
`som
`re
`
`+
`
`2\b
`
`Ilb
`
`|
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 6
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 5 of 12
`
`6,159,739
`
`vr
`Ww
`
`co
`
`rm
`._ WO
`
`w
`
`a)
`uw)
`
`®
`s
`a
`
`—
`t
`
`Q
`©
`
`IIb
`
`O
`lo
`
`®
`_
`=
`
`oO
`T
`
`&
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 7
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 6 of 12
`
`6,159,739
`
`llc
`
`Alc
`
`8b
`
`Figure 6
`
`ABS Global, Inc. and Genusple — Ex. 1005, p. 8
`
`

`

`U.S. Patent
`
`Dec.12, 2000
`
`Sheet 7 of 12
`
`6,159,739
`
`s F
`
`igure 7A
`
`q F
`
`igure 7B
`
`d F
`
`igure 7C
`
`Figure 7D
`
`ABSGlobal, Inc. and Genus ple — Ex. 1005, p. 9
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 8 of 12
`
`6,159,739
`
`l2
`
`a:A
`
`errnOOO
`
`—___
`
`ee.
`
`Figure 8A
`
`30
`
`Frequency
`
`20
`
`oO
`
`O
`
`25
`
`45
`
`65 8 10
`
`Velocity (micron/sec)
`
`Figure 8B
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 10
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 9 of 12
`
`6,159,739
`
`Figure 9
`
`ABS Global, Inc. and Genusple — Ex. 1005, p. 11
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 10 of 12
`
`6,159,739
`
`ae
`
`4{d
`
`ld
`
`80
`
`90
`TO
`
`TI
`
`9]
`
`Figure
`
`IOA
`
`8]
`
`oan
`
`v4)
`
`Figure |IOB
`
`ABS Global, Inc. and Genusple — Ex. 1005, p. 12
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 11 of 12
`
`6,159,739
`
`70
`
`Se
`
`eal
`
`vai
`
`Figure |IOD
`
`
`
`Figure |OC
`
`ABSGlobal, Inc. and Genus ple — Ex. 1005, p. 13
`
`

`

`U.S. Patent
`
`Dec. 12, 2000
`
`Sheet 12 of 12
`
`6,159,739
`
`lOf
`
`oA
`
`oy
`
`lOO
`
`5
`
`uf
`
`7f
`
`if
`
`Sf
`
`30f
`
`Figure IlA
`
` 5
`
`out
`
`vf
`
`elf
`
`St
`
`Figure IIB
`
`ABS Global, Inc. and Genus ple — Ex. 1005, p. 14
`
`

`

`6,159,739
`
`1
`DEVICE AND METHODFOR3-
`DIMENSIONAL ALIGNMENT OF
`PARTICLES IN MICROFABRICATED FLOW
`CHANNELS
`
`This invention was made with government support. The
`government has certain rights therein.
`FIELD OF INVENTION
`
`This invention relates generally to an apparatus and
`methods for achieving sheath flow using a microfabricated
`flow channel. The invention is useful, for example, for
`injecting small particles into a sheath stream, for achieving
`sheath flow in a flow cytometer and in creating hydrody-
`namic focusing.
`BACKGROUND OF THE INVENTION
`
`Flow cytometry is a sensitive and versatile probe of the
`optical characteristics of microscopic particles, with wide-
`spread applications including hematology,
`immunology,
`genetics,
`food science, pharmacology, microbiology,
`parasitology, and oncology. Optical flow cytometers use
`light scattering and fluorescence to determine physical and
`chemical properties of the particles. For measurement, par-
`ticles are arranged in singlefile, typically by hydrodynamic
`focusing within a sheath fluid, and interrogated by a light
`beampropagating orthogonal to the flow axis. Flow cytom-
`eters often use two concentric fluids to carry particles
`through the measurement zone, where optical measurement
`occurs. The use of two concentric fluids facilitates the
`passage of the particles through the measurement zone in a
`single file fashion, and helps avoid clogging of the flow
`channel. Hydrodynamic focusing is a phenomenon that
`leads to a single file flow of particles as a result of the very
`small dimensions of the flow channel. A sample is injected
`into a flowing sheath fluid;
`the dimensions of the flow
`channel become more narrow, causing the dimensionsof the
`stream of sample to become more narrow also. FIG. 1 is a
`cross section of flow in an art-known sheath flow accom-
`
`plished by injecting, via a needle or other concentric
`opening, a center fluid (41) containing a sample with par-
`ticles (42)
`into a sheath fluid (40), surrounded by air.
`Hydrodynamic focusing requires laminarflow of the fluids;
`any turbulence would cause mixing of the concentric fluids.
`The optical properties of the particles are measured in the
`measurement zone. Scattered light and fluorescence are
`measured by two or more photodetectors positioned around
`the illuminated portion of the flow stream. A first photode-
`tector can be positioned to collect small angle scattering. A
`second photodetectoris often positioned at about 90° to the
`forward scattering direction to collect large angle scattering
`and fluorescence.
`
`Existing commercial cytometers are large and compli-
`cated instruments requiring skilled operators. To increase the
`accessibility of flow cytometry, compact cytometers are
`desired.
`
`The flow behavior of liquids at the microscopic level is
`significantly different from the flow behavior of liquids at
`the macroscopic level. In microstructures, i.e. microfabri-
`cated fluidic devices, practically all flow is laminar, as a
`result of the extremely small channel diameters. Laminar
`flow allows two or morefluids to flow parallel to each other
`without turbulence-induced mixing. However, because the
`channel diameters are very small, diffusion is significant.
`Since diffusion occursin all directions, a component of one
`layer may diffuse to another layer, perpendicular to the
`direction of flow.
`
`10
`
`15
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`Sheath flow ts a particular type of laminar flow in which
`one layer is surrounded by another layer on more than one
`side. Concentric layers of fluids, that is, where one layer is
`completely surrounded onall sides by another layer, is one
`example of sheath flow. Sheath flow is useful because it
`positions particles with respect to illuminating light, e.g., a
`laser beam, and it prevents particles 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 allowsfor faster flowvelocities and
`
`higher through-put of sample material. Faster flow velocity
`is possible without shredding cells in the center fluid
`because the sheath fluid protects the cells from shear forces
`at the walls of the flow channel. Sheath flow is useful in
`many applications, including but not limited to, any appli-
`cation in which it is preferable to protect particles by a layer
`of fluid, for example in applications wherein it is necessary
`to protect particles from air. Other applications include flow
`cytometry and combustion processes wherein an inner core
`layer burnsat a different temperature from that of an outer
`layer. In the latter application, the sheath flow moduleofthis
`invention can be used to create a flame of two or more
`
`combustible fluids with the outer sheath fluid burning, for
`example, at a higher temperature than the inner core fluid. In
`this case, the outer sheath fluid can be used to heat the inner
`core fluid. Of course, the temperatures of the fluids can be
`reversed,i.e., the outer sheath fluid can be one which burns
`at a lower temperature than the inner core fluid. Control of
`flame shape or color is possible using the sheath flow
`module of this invention.
`
`In a microfabricated flow channel, a challengeis to focus
`light into the channel and to collect near forward scattered
`and high angled scattered light out of the channel. A few
`microfabricated flow cytometer flow channels have been
`reported. Miyake et al. [Proceedings of the IEEE Micro
`Electro Mechanical Systems Workshop, pp. 265-270, Nara,
`Japan, January 1991] describes a micromachined sheath
`flow channel madeoffive stacked plates. Three metal plates
`are used to create a flow having a sample core within a
`sheath, and glass plates on the top and bottom of the stack
`provide optical access to the flow channel for illumination
`through the top and forward scattered light collection
`through the bottom. The top and bottom plates provide a
`sheath fluid inlet. The middle plate provides for the sample
`inlet in the center, with the sheath fluid inlets on both sides.
`It appears that 90° scattering cannot be collected. Sobek et
`al. [Proceedings of the IEEE Micro Electro Mechanical
`Systems Workshop, pp. 219-224, [ort Lauderdale, ['la.,
`February 1993] describes a four-layer silicon microfabri-
`cated hexagonal sheath flow channel. The channelis formed
`between two of the silicon wafers.
`Integrated optical
`waveguides intersecting the channel are used to couple laser
`light into the channel and out of the channel in the forward
`direction. At this intersection, the top and bottom walls of
`the channel are silicon nitride/silicon dioxide windowsfor
`90° light collection. Each windowis fabricated by growing
`an oxide layer on a silicon wafer, bonding the oxide layer to
`a second silicon wafer, etching away the silicon on both
`sides of the oxide at the window region and depositing a
`nitride layer. Sheath flow with a sample in the center of the
`sheath stream is accomplished by injecting sample via a
`hypodermic needle into the center of the stream of sheath
`fluid. Sobek et al. [Proceedings of the Solid-State Sensors
`and Actuators Workshop, Hilton Head, S.C., June 1994]
`describes a sheath flow channel fabricated between two
`fused silica wafers. To couple light into the channel and out
`in the forward direction, optical fibers are sandwiched
`
`(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:24)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:24)
`ABS Global, Inc. and Genusplc — Ex. 1005, p. 15
`
`

`

`6,159,739
`
`3
`between the wafers orthogonal to the flow axis. Fluores-
`cence is collected through the upper transparent water.
`Again, sheath flow is accomplished by injection of the
`sample via a hypodermic needle into the center of the sheath
`stream.
`
`US. Pat. No. 5,726,751, “Silicon Microchannel Optical
`Flow Cytometer,” issued Mar. 10, 1998, which is incorpo-
`rated by reference herein in its entirety, discloses a flow
`cytometer comprising a v-groove flow channel formed by
`micromachining a silicon wafer. This reference describes a
`flow cytometer made of two components: a flow cytometer
`optical head and a disposable flow module. The flow module
`of this reference exploits the fact that anisotropic etching of
`single crystalline silicon wafers provides access to reflective
`surfaces with precisely etched anglesrelative to the surface
`of the wafer. The facets are used for reflecting, as opposed
`to transmitting, an illuminating laser beam. This reference
`suggests the use of a sheath flow in a v-groove but does not
`teach a novel method or apparatus for achieving sheathflow.
`SUMMARYOF THE INVENTION
`
`10
`
`15
`
`4
`junction, into an established sheath fluid. Unlike the previ-
`ously known devices which create sheath flow (e.g. Miyake
`et al.), the present invention does not need sheath inlets on
`eachside of the flow channel. Optionally, to get sheath fluid
`above and below the center fluid so that the center fluid is
`
`entirely surrounded by sheath fluid, a third inlet can be
`provided for introduction of sheath fluid downstream of the
`center fluid (second) inlet. The sheath flow module may be
`oriented in any way, e.g., horizontally, vertically, or tilted
`(with respect to the long axis of the flow channel).
`In this mode the inlets may be on the bottom ortop of the
`flow channel. This results in sheath fluid on two sides and
`
`either the top or the bottom, respectively. If the module is
`oriented with the inlets on the bottom of the flow channel,
`then upon introduction of the center fluid, sheath flow is
`accomplished with the center fluid surrounded by the sheath
`fluid on the top and onthe sides. Alternatively, if the module
`is oriented with the inlets on the top of the flow channel, then
`upon introduction of the center fluid, sheath flow is accom-
`plished with the center fluid surrounded bythe sheath fluid
`on the bottom and on the sides.
`
`30
`
`To surround the center fluid on all sides, the sheath flow
`The present invention provides a sheath flow module
`made fromafirst plate, which is a single piece of material,
`module of the present
`invention may further comprise
`and a second plate, which is preferably a transparent cover
`additional inlets, for instance, a third inlet, downstream of
`plate. This module allows for sheath flow creation and
`the secondinlet. The third inlet may be used to introduce any
`optical measurements of a sample of interest. The sheath
`desired fluid. For example, a third inlet may be included to
`flow module of the present invention can be employed to
`introduce a second layer of sheath fluid so that the center
`achieve sheath flow on a microscale for use in flow cytom-
`fluid is surrounded on all sides by the sheath fluid. If the
`etry. An object of the present invention is to provide a flow
`third inlet is used for introducing sheath fluid, the third inlet
`module for reproducibly focusing particles into the mea-
`is preferably wider than the second inlet, more preferably
`surement zone of a flow cytometer. Another object of the
`approximately the same width asthefirst inlet. Alternatively,
`invention is to prevent particles from touching the walls/
`a third or additional inlet may be included to, for example,
`sides and bottom and top of the flow channel.
`introduce a reagent which chemically reacts with or other-
`wise modifies a sample already introduced.
`The present invention provides a sheath flow module
`35
`The laminar flow channelof the sheath flow module of the
`made fromafirst plate of material having formed therein a
`laminar fluid flow channel; at least two inlets, each inlet
`present invention can increase in depth at any and/or each
`joining the laminar flow channel at a junction, the first inlet
`inlet. For example, the depth of the channel may be greater
`junction being wider than the second inlet junction, and an
`between the second inlet junction and the outlet than the
`outlet from the flow channel. A second plate, e.g. a trans-
`depth between the first and second inlet junctions. If a third
`parent cover plate, seals the module and allows for optical
`inlet is present, the depth of the channel between the second
`measurements.
`and third inlet junctions may be greater than the depth
`betweenthe first and second inlet junctions but less than the
`depth between the third inlet junction and the outlet. An
`increase in depth provides for introduction of an additional
`fluid layer with retention ofall fluid layer dimensions(layers
`already flowing in the channel, as well as the newly injected
`layer). This retention of all
`fluid layer dimensions is
`preferable, but not necessary.
`Afterall fluids have been introduced and sheath flow has
`been achieved,
`the depth of the flow channel can be
`decreased,
`leading to vertical hydrodynamic focusing.
`Optionally, the width of the flow channel can be decreased,
`leading to horizontal hydrodynamic focusing. The decrease
`in depth and width can be gradual or abrupt.
`The second mode, termed the particle injector mode, is
`useful in cases in which the center fluid contains particles
`which are denser or less dense than the fluids surrounding
`them.
`In the present
`invention, one or both fluids may
`contain particles. If the particles are denser than the fluids
`surrounding them,
`the particles settle out of the fluids
`(gravity pulls the particles down), and hence the particles
`can elude measurement. In the present invention, this prob-
`lemofparticles settling out of the fluids as a result of gravity
`can be avoided byorienting the sheath flow module verti-
`cally. Alternatively, gravity can be exploited by orienting the
`module horizontally and positioning the inlets on top of the
`
`The present invention providesthe creation of sheath flow
`from a single plate with formed features, covered by a
`second plate. The second plate can either be a flat cover
`plate, preferably transparent, or it can have a channel and/or
`inlets and/or an outlet formed therein. A transparent cover
`plate allows for optical measurements byreflection, in cases
`where the first plate is a reflective material, e.g. silicon. In
`cases where the first and second plates are both transparent,
`optical measurements can be performed by transmission.
`There is no need to fabricate and align multiple plates or use
`a syringe needle for injection.
`A first inlet allowsfor introductionofa first fluid into the
`flow channel. Thefirstfluid is the sheath fluid. Asecond inlet
`allows for introduction of a second fluid into the sheath fluid
`
`while it is flowing through the flow channel. The second
`fluid is the center fluid. Because the second inlet junction is
`narrower than the first
`inlet junction,
`the center
`fluid
`becomes surrounded on both sides by the sheath fluid.
`The device of the present invention can function in two
`modes, the sheath flow mode andthe particle injector mode,
`depending on the relative densities of the sheath fluid, the
`center fluid, and any particles in either fluid.
`Thefirst mode, termed the sheath flow mode, is for fluids
`and particles of approximately equal densities, and creates
`sheath flow by introducing a center fluid, via a narrow inlet
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`(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:24)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:25)
`ABS Global, Inc. and Genusplc — Ex. 1005, p. 16
`
`

`

`6,159,739
`
`5
`module. In this mode, particles are injected by first estab-
`lishing a flow of sheath fluid whichis introducedviathefirst
`inlet, and then introducing particles or particle-containing
`center fluid via the second inlet. Because the particles are
`denser than the center and sheath fluids, gravity acts to
`center the particles in the vertical dimension. Thus,
`the
`second mode of this invention provides for deliberate mix
`(as a result of gravity) of a center fluid constituent with
`sheath fluid (which was avoided in the first mode) and
`allows the particles to be surrounded onall sides by sheath
`fluid (which was accomplished by a third inlet in the first
`mode).
`If the particles are less dense than the surroundingfluids,
`they can be injected from the bottom, from whencethey float
`upwardas a result of their buoyancy.If the particles are less
`dense than the surrounding fluids, it may be preferable to
`introduce a second sheath fluid,via a third inlet, especially
`if the particles are injected from the top.
`In the second mode, the injected particles can be fluores-
`cent beads. It is preferable to have the inlets on the top ofthe
`flow channel in this mode. Thus, gravity causes the particles
`to fall slowly as the fluids flow through the channel. US.
`Pat. No. 5,747,349 issued Mar. 5, 1998, “Fluorescent
`Reporter Beads for Fluid Analysis,” which is incorporated
`by reference herein in its entirety, discloses reporter beads
`for chemical analysis of fluid properties such as pH, oxygen
`saturation and ion content. A fluorescent property of the
`reporter bead, such as intensity, lifetime or wavelength, is
`sensitive to a corresponding analyte. Reporter beads are
`added to a fluid sample and the analyte concentration is
`determined by measuring the fluorescence of individual
`beads. Beads tagged with different reporter molecules allow
`for a plurality of analytes in a sample to be measured
`simultaneously. Alternatively, absorptive beads tagged with
`reporter molecules which change absorbance as a function
`of analyte concentration can be employed in a manner
`similar to the fluorescent beads.
`For use in either mode, the module can include a mea-
`surement zone between the most downstream inlet and the
`
`outlet. The measurement zone provides optical access for
`measurements such as scattering, absorbance, fluorescence
`and emission. The distance between an inlet through which
`particles are introduced and the outlet is preferably chosen
`such that, for a given flow speed, the particles do not touch
`the bottom or top of the flow channel. In the particle injector
`mode,the measurementzoneis preferably positioned so that
`the particles are surrounded on all sides byfluid and have not
`dropped so close to the bottom or floated so close to the top
`of the flow channel that optical measurements are hindered.
`In a preferred embodiment, optical measurements exploit
`reflection from the channel walls, as described in US. Pat.
`No. 5,726,751 “Silicon Microchannel Optical Flow
`Cytometer,” issued Mar. 10, 1998.
`Prior to the measurement zone, the diameter of the flow
`channel can taper from a wider to a narrower downstream
`portion. This narrowing of the channel leads to horizontal
`hydrodynamic focusing.
`The sheath flow module of this invention can be applied
`to many systems. For example, the sheath fluid may be a
`sample fluid and the particles in the center fluid may be
`reporter beads. Alternatively, the sheath fluid may beaninert
`fluid and the center fluid may be a sample,e.g., blood. These
`are just a few of the many types of systems in which the
`present invention can be applied.
`The sheath flow module may be made from anyetchable,
`machinable or moldable material, e.g. silicon wafers,
`plastics, and casting materials.
`
`6
`This invention further provides methods for focusing a
`centerfluid in a sheath fluid using the sheath flow module of
`the present invention. The methods include introducing a
`sheath fluid into the first inlet; introducing a center fluid into
`the second inlet, and optionally introducing a third fluid
`(which maybe sheath fluid) into an optionalthird inlet. The
`center fluid may be a sample fluid, and/or may contain
`reporter beads. The sheath fluid may be a particle-free
`carrier fluid (optically inert) or it may contain particles. The
`sheath fluid may be a sample fluid, e.g. whole blood.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a cross section of sheath flow achieved by a
`known method.
`
`15
`
`FIG. 2 is a cross section of a v-groove flow channel.
`FIG. 3A is a lengthwise section through the center of the
`sheath flow module of this invention.
`
`FIG. 3B is a top view of the sheath flow module of this
`invention.
`FIG. 3C is a crass section of the flaw channel of FIGS. 3A
`and 3B and sheath flow attained therein.
`
`FIG. 4A is a lengthwise section of a particle injector,
`showing falling particles.
`FIG. 4B is a top view of a particle injector, showing the
`relative widths of inlets and portions of the laminar flow
`channel.
`
`FIG. 5A is a lengthwise section through the center of a
`sheath flow flow cytometer.
`FIG. 5B is a top view of the sheath flow flow cytometer
`of FIG. 5A.
`
`FIG. 5C is a lengthwise section through the flow channel
`some distance from the center, i.e. not through the second
`inlet, in the flow channel of FIGS. 5A and 5B in whichthe
`depth of the channel increasesat the second inlet only across
`the width of the second inlet and increases across the entire
`width of the channel at the third inlet.
`
`FIG. 5D is a cross section ofthe fluid flowing in the flow
`channel of FIGS. 5A, B and C.
`FIG. 6 is a lengthwise section through the flow channel
`some distance from the center, i.e. not through the second
`inlet, in an alternative embodiment to the flow channel of
`FIGS. 5A-5D.
`
`FIG. 7, comprising FIGS. 7A—7D, showscross sections of
`various embodiments of the flow channel of this invention.
`
`FIG. 8A showstherelative velocities of the various layers
`of fluids in the laminar flow channel.
`
`FIG. 8B is a velocity histogram of fluorescent beads in a
`sheath flow channel. Frequency (a measure of the number of
`beads) versus velocity of the beads is graphed.
`FIG. 9 illustrates the channels of an embodiment of the
`
`“H” filter of U.S. Pat. No. 5,932,100 issued Aug. 3, 1999.
`FIG. 10A illustrates a sheath flow module, having a
`branching flow channel.
`FIG. 10B illustrates the use of a branching flow channel
`to divide the fluid flowing through the flow channel.
`FIG. 10C shows the branching flow channels of a sheath
`flow module.
`
`FIG. 10D shows that the branching flow channels may
`join the flow channel at angles other than 90 degrees.
`FIG. 11A showsthe sheath flow module creating a flame.
`FIG. 11B showsthe sheath flow module with an outlet at
`the downstream end of the channel for introducing particles
`into a flame for emission spectroscopy.
`
`40
`
`50
`
`55
`
`60
`
`65
`
`(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:24)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:26)
`ABS Global, Inc. and Genusplc — Ex. 1005, p. 17
`
`

`

`6,159,739
`
`7
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`‘The sheath flow module of the present invention provides
`that a center stream is surrounded on morethan one side and
`
`optionally on all sides by a sheath fluid. This provides a way
`to position a particle in the channel. This helps prevent
`clogging of the flow channel and provides a uniform speed
`of the particle in the channel. Precise positioning and
`uniform speed of particles are generally useful in detection
`schemes,e.g., in flow cytometry.
`The term sheath fluid refers to a layer of fluid, which may
`contain particles, surrounding on more than oneside a center
`fluid. The term center fluid refers to a layer of fluid, which
`may contain particles, which is surrounded on more than one
`side bya sheath fluid.
`The sheath fluid can be an optically and chemically inert
`fluid or it can be a sample containing cells and/or analytes
`of interest.
`
`The center fluid can be a sample fluid which can contain
`fluorescent reporter beads, and/or cells and/or analytes of
`interest and/or a chemical indicator and/or a reagent which
`reacts with an analyte of interest to give a changein detected
`optical properties. The center fluid can be a non-sample fluid
`which contains reporter beads, an indicator or a reagent.
`‘The term particles has the common meaning,andrefers to
`undissolved solid matter, and specifically includes cells and
`fluorescent beads.
`
`FIG. 2 is a cross section of a v-groove flow channel. A
`v-groove (56) is etched in a silicon wafer (43) and the
`channel is sealed by a transparent, e.g. glass, cover plate
`(44). A particle (42) is surrounded by sample fluid (41).
`The sheath flow module of this inventionis illustrated in
`
`FIGS. 3A, 3B and 3C. FIG. 3A is a lengthwise section
`through the center of the flow module. Plate (1) is machined,
`molded or etched to form the flow channel. The plate can be
`selected from the following which include, but are not
`limited to, silicon wafers, plastics, e.g. polypropylene, and
`casting materials. Techniques for etching silicon wafers and
`molding and machining plastics are well-known in theart.
`The plate has at least one surface (6), which is a substan-
`tially flat plane. A laminar flow channel (8) is formedinflat
`plane of the plate. The term laminar flow channel is used
`herein to refer to a flow channel having dimensions that
`a