(12) United States Patent
`Wada et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006506609Bl
`US 6,506,609 Bl
`Jan.14,2003
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) FOCUSING OF MICROPARTICLES IN
`MICROFLUIDIC SYSTEMS
`
`(75)
`
`Inventors: H. Garrett Wada, Atherton, CA (US);
`Anne R. Kopf-Sill, Portola Valley, CA
`(US); Marja Liisa Alajoki, Palo Alto,
`CA (US); J. Wallace Parce, Palo Alto,
`CA (US); Benjamin N. Wang, Palo
`Alto, CA (US); Andrea W. Chow, Los
`Altos, CA (US); Robert S. Dubrow,
`San Carlos, CA (US)
`
`(73) Assignee: Caliper Technologies Corp., Mountain
`View, CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/569,747
`
`(22) Filed:
`
`May 11,2000
`
`Related U.S. Application Data
`( 60) Provisional application No. 60/134,472, filed on May 17,
`1999.
`
`Int. Cl.7
`(51)
`(52) U.S. Cl .
`
`.................................................. GOlN 7/00
`........................... 436/148; 436/34; 436/52;
`436/180; 436/518; 422/50; 435/91.1
`(58) Field of Search ............................ 436/148, 34, 52,
`436/180, 518; 422/50; 204/452, 454, 600;
`356!73; 435/7.1, 6, 287.3, 91.1; 210/634
`
`(56)
`
`References Cited
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`
`Primary Examiner-Jill Warden
`Assistant Examiner-Brian Sines
`(74) Attorney, Agent, or Finn-Andrew L. Filler
`
`(57)
`
`ABSTRACT
`
`Methods and systems for particle focusing to increase assay
`throughput in microscale systems are provided. The inven(cid:173)
`tion includes methods for providing substantially uniform
`flow velocity to flowing particles in microfluidic devices.
`Methods of sorting members of particle populations, such as
`cells and various subcellular components are also provided.
`Integrated systems in which particles are focused and/or
`sorted are additionally included.
`
`35 Claims, 22 Drawing Sheets
`
`16
`
`

`
`US 6,506,609 Bl
`Page 2
`
`U.S. PATENT DOCUMENTS
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`
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`........... 204/452
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`wo 98/02728
`wo 98/05424
`wo 98/22811
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`
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`
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`wo
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`wo
`wo
`wo
`wo
`wo
`wo
`
`wo 98/46438
`wo 98/49548
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`wo 98/56956
`wo 99/00649
`wo 99/10735
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`wo 99!16162
`wo 99/19056
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`
`10/1998
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`OTHER PUBLICATIONS
`
`Jacobson, S.C. et al., "Fused Quartz Substrates for Micro(cid:173)
`chip Electrophoresis," A.nal. Chern. (1995) 67:2059-2063.
`Kessler J., "Hydrodynamic focusing of motile algal cells"
`Nature vol. 313 pp. 218-220.
`Knight J., et al., "Hydrodynamic Focusing on a Silicn Chip:
`Mixing Nanoliters in Microseconds" Physical Review Let(cid:173)
`ters (1998) vol. 80, No. 17 pp. 3863-3866.
`Kononenko and Shimkus "Non-equilibrium integral Dop(cid:173)
`pler anemometric analysis of particel mixtures in a channel
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`517-530.
`Manz, A. et al., "Electroosmotic pumping and electro(cid:173)
`phoretic separations for miniaturized chemical analysis sys(cid:173)
`tems," J. Micromech. Microeng. (1994) 4:257-265.
`Ramsey, J.M. et al., "Microfabricated chemical measure(cid:173)
`ment systems," Nature lvled. (1995) 1:1093-1096.
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`phoresis: Repetitive Sample Injection, Quantitation, and
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`Seiler, K. et al., "Electroosmotic Pumping and Valveless
`Control of Fluid Flow Within a Manifold of Capillaries on
`a Glass Chip," A.nal. Chern. (1994) 66:3485-3491.
`Sundberg, S. A., "High-throughput and ultra-high-through(cid:173)
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`etry" Cytometry (1999) vol. 38 pp. 2-14.
`
`* cited by examiner
`
`wo
`wo
`wo
`wo
`wo
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`
`

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`U.S. Patent
`
`Jan.14,2003
`
`Sheet 1 of 22
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`US 6,506,609 Bl
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`
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`

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`U.S. Patent
`
`Jan.14,2003
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`Jan.14,2003
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`U.S. Patent
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`Jan.14,2003
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`
`

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`U.S. Patent
`
`Jan.14,2003
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`Sheet 12 of 22
`
`US 6,506,609 Bl
`
`- - - -
`
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`
`

`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 13 of 22
`
`US 6,506,609 Bl
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`L()
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`

`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 14 of 22
`
`US 6,506,609 Bl
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`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 15 of 22
`
`US 6,506,609 Bl
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`

`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 16 of 22
`
`US 6,506,609 Bl
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`
`Jan.14,2003
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`U.S. Patent
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`Jan.14,2003
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`Sheet 18 of 22
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`Jan.14,2003
`
`Sheet 19 of 22
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`US 6,506,609 Bl
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`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 20 of 22
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`US 6,506,609 Bl
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`

`
`U.S. Patent
`
`Jan.14,2003
`
`Sheet 21 of 22
`
`US 6,506,609 Bl
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`

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

`
`US 6,506,609 Bl
`
`1
`FOCUSING OF MICROPARTICLES IN
`MICROFLUIDIC SYSTEMS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application is related to and claims priority to and the
`benefit of provisional application 60/134,472, filed May 17,
`1999, Wada et al., "Focusing of Microparticles in Microf(cid:173)
`luidic Systems," pursuant to 35 U.S.C. §119(e), as well as
`any other applicable statute or rule. This priority application
`is incorporated herein in its entirety for all purposes.
`
`COPYRIGHT NOTIFICATION
`
`Pursuant to 37 C.P.R. 1.71(e), Applicants note that a
`portion of this disclosure contains material which is subject
`to copyright protection. The copyright owner has no objec(cid:173)
`tion to the facsimile reproduction by anyone of the patent
`document or patent disclosure, as it appears in the Patent and
`Trademark Office patent file or records, but otherwise
`reserves all copyright rights whatsoever.
`
`BACKGROUND OF THE INVENTION
`
`s
`
`2
`washed away. Other well-known sorting methods include
`those using fluorescence-activated cell sorters ("FACSs").
`FACSs for use in sorting cells and certain subcellular
`components such as molecules of DNA have been proposed
`in, e.g., Fu, A. Y. et al. (1999) "A Microfabricated
`Fluorescence-Activated Cell Sorter," Nat. Biotechnol.
`17:1109-1111; Unger, M., et al. (1999) ''Single Molecule
`Fluorescence Observed with Mercury Lamp Ilumination,"
`Biotechniques 27:1008-1013; and Chou, H. P. et al. (1999)
`10 "A Microfabricated Device for Sizing and Sorting DNA
`Molecules," Proc. Nat'!. Acad. Sci. 96:11-13. These sorting
`techniques utilizing generally involve focusing cells or other
`particles by flow channel geometry.
`While cell-based assays are generally preferred in certain
`15 microscale screening applications, certain of these assays
`are difficult to adapt to conventional notions of high(cid:173)
`throughput or ultra high-throughput screening assay sys(cid:173)
`tems. For example, one difficulty in flowing assay systems
`is that, during pressure-based flow of fluids in channels,
`20 non-uniform flow velocities are experienced. Faster fluid
`and material flow is observed in the center of a moving fluid
`stream than on the edge of a moving fluid stream. This
`non-uniform flow velocity reduces throughput for flowing
`assays, because assay runs have to be spaced well apart in
`25 the fluid stream to prevent overlap of materials moving at
`different velocities.
`Accordingly, it would be advantageous to provide mecha(cid:173)
`nisms for facilitating cell-based assays, including cell sort(cid:173)
`ing techniques, especially in microscale systems. Additional
`microscale assays directed at subcellular components, such
`as nucleic acids would also be desirable. The present inven(cid:173)
`tion provides these and other features which will become
`clear upon consideration of the following.
`
`A variety of cell-based assays are of considerable com(cid:173)
`mercial relevance in screening for modulators of cell-based
`activity. For example, compounds which affect cell death
`can have profound biological activities and are desirably
`screened for in cell-based assays. Cell death has become
`recognized as a physiological process important in normal 30
`development, hormonal regulation of various tissues, and,
`e.g., in regulation of the receptor repertoires of both T and
`B lymphocytes. The finding that a pattern of morphological
`changes is common to many examples of programmed cell
`death (or PCD) led to the suggestion of a common 35
`mechanism, and the term "apoptosis" was defined to include
`both the morphological features and the mechanism com(cid:173)
`mon to such programmed cell death (Kerr et al., Br. J.
`Cancer 26:239). This concept was extended by the finding
`that nuclear DNA fragmentation correlates well with apop- 40
`totic morphology (Arends et al., Am. J. Pathol. 136:593
`(1990)), and the scientific literature contains many examples
`of PCD accompanied by these features. There are also clear
`examples of PCD in the absence of apoptotic morphology or
`DNA fragmentation (Clarke,Anat. Embryl. 181:195 (1990), 45
`Martinet al,J. Cell Bioi. 106:829 (1988), and Ishigami et al.,
`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 50
`effect(s) on apoptosis or other biological phenomena. Pio(cid:173)
`neering technology providing cell- and other particle-based
`microscale assays are set forth in Parce et al. "High
`Throughput Screening Assay Systems in Microscale Fluidic
`Devices" WO 98/00231; in PCT;US00/04522, filed Feb. 22, 55
`2000, entitled "Manipulation of Microparticles In Microf(cid:173)
`luidic Systems," by Mehta et al.; and in PCTUS00/04486,
`filed Feb. 22, 2000, entitled "Devices and Systems for
`Sequencing by Synthesis," by Mehta et al.
`Other cell-based assays include various methods for the
`preparative or analytic sorting of different types of cells. For
`example, cell panning generally involves attaching an
`appropriate antibody or other cell-specific reagent to a solid
`support and then exposing the solid support to a heteroge(cid:173)
`neous cell sample. Cells possessing, e.g., the corresponding
`membrane-bound antigen will bind to the support, leaving
`those lacking the appropriate antigenic determinant to be
`
`SUMMARY OF THE INVENTION
`
`The present invention relates to methods of focusing
`particles in microchannels, e.g., to improve assay
`throughput, to sort particles, to count particles, or the like. In
`the methods of the invention, cells and other particles are
`focused in the center of, to one side of, or in other selected
`regions of microscale channels, thereby avoiding, e.g., the
`above noted difficulties inherent in pressure-based flow of
`particles. Furthermore, the device structures of the present
`invention are optionally integrated with other microfluidic
`systems. Other reactions or manipulations involving cells,
`other particles, or fluids upstream of the detection zone are
`also optionally performed, e.g., monitoring drug interactions
`with cells or other particles.
`In one aspect, the invention provides methods of provid(cid:173)
`ing substantially uniform flow velocity to particles flowing
`in a first microchannel. In the methods, the particles are
`optionally flowed in the microchannel, e.g., using pressure(cid:173)
`based flow, in which the particles flow with a substantially
`non-uniform flow velocity. Prior to performing the flowing
`step, the particles are optionally sampled with at least one
`capillary element, e.g., by dipping the capillary element into
`a well containing the particles on a microwell plate and
`drawing the particles into, e.g., reservoirs, microchannels, or
`60 other chambers of the device. The particles (e.g., a cell, a set
`of cells, a microbead, a set of microbeads, a functionalized
`microbead, a set of functionalized microbeads, a molecule,
`a set of molecules, etc.) are optionally focused horizontally
`and/or vertically in the first microchannel to provide sub-
`65 stantially uniform flow velocity to the particles in the first
`microchannel. Particles are optionally focused using one or
`more fluid direction components (e.g., a fluid pressure force
`
`

`
`US 6,506,609 Bl
`
`5
`
`3
`modulator an electrokinetic force modulator, a capillary
`force modulator, a fluid wicking element, or the like).
`Additional options include sorting, detecting or otherwise
`manipulating the focused particles.
`The particles are horizontally focused in the
`microchannel, e.g., by introducing a low density fluid and a
`high density fluid into the microchannel, causing the par(cid:173)
`ticles 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
`densitv fluid into the microchannel with the flowing par(cid:173)
`ticles.,The particles are vertically or horizontally focused in
`the microchannel, e.g., by simultaneously introducing fluid
`flow from two opposing microchannels into the first micro(cid:173)
`channel during flow of the particles in the first channel.
`Vertical focusing is also optionally achieved to one side of
`a microchannel by simultaneously introducing fluid flow
`from, e.g., a second microchannel into the first microchannel
`during flow of the particles in the first microchannel.
`In another aspect, the invention also provides particle
`washing or exchange techniques. For example, focused cells
`or other particles are optionally washed free of diffusible
`material by introducing a diluent into the first microchannel
`from at least a second channel and removing the resulting 25
`diluted diffused product comprising diluent mixed with the
`diffusible material through at least a third microchannel.
`Alternating arrangements of diluent input and diffused
`product output channels are also optionally used to further
`wash the particles. For example, in one aspect the methods
`of the invention include simultaneously introducing the
`diluent into the first microchannel from the second micro(cid:173)
`channel and a fourth microchannel, where the second and
`fourth microchannel intersect the first microchannel at a
`common intersection region. Optionally, the methods
`include sequentially introducing the diluent into the first
`microchannel from the second microchannel and a fourth
`microchannel, wherein the second and fourth microchannels
`intersect the first microchannel at an offset intersection
`region. The diffused product is typically removed through
`the third microchannel and a fifth microchannel, which third
`and fifth microchannels intersect the first microchannel at a
`common intersection region. In further washing steps, the
`diluent is introduced through sixth and seventh microchan(cid:173)
`nels which intersect the first microchannel at a common
`intersection. The resulting further diluted diffused product is
`removed through eighth and ninth microchannels, which
`intersect the first microchannel at a common intersection.
`Diluent is optionally introduced into the first microchannel
`by pressure or electrokinetic flow.
`In one preferred assay of the invention, the particles are
`cells and the method includes performing a TUNEL assay or
`an Annexin-V assay on the cells in the channel to measure
`apoptosis.
`Integrated systems for performing the above methods,
`including the particle sorting embodiments, are also pro(cid:173)
`vided.
`An integrated system for providing substantially uniform
`flow velocity to flowing members of at least one particle
`population in a microfluidic device optionally includes a
`body structure that includes at least a first microchannel
`disposed therein. A first fluid direction component (e.g., a
`fluid pressure force modulator) is typically coupled to the
`first microchannel for inducing flow of a fluidic material that
`includes the members of the at least one particle population
`in the first microchannel. The first fluid direction component
`
`4
`generally induces non-uniform flow. A source of at least one
`fluidic material is optionally fluidly coupled to the first
`microchannel. The system also optionally includes at least a
`second microchannel that intersects the first microchannel
`for introducing at least one fluid into the first microchannel
`to horizontally or vertically focus the members of the at least
`one particle p~pulation in the first microchannel. The at least
`one fluid is optionally introduced using a second fluid
`direction component that includes one or more of a fluid
`pressure force modulator, an electrokinetic force modulator,
`10 a capillary force modulator, a fluid wicking element, or the
`like. At least one flow control regulator for regulating flow
`of the fluidic material or the fluid in the first or second
`microchannel is also optionally provided. A computer
`including an instruction set directing simultaneous flow of
`15 the members of the at least one particle population in the first
`microchannel and simultaneous introduction of the at least
`one fluid from the second microchannel into the first micro(cid:173)
`channel is optionally also operably coupled to a fluid move(cid:173)
`ment system for directing flow of materials in the micro-
`20 channels.
`As a further option, this integrated system additionally
`includes at least a third microchannel which intersects the
`first microchannel in an intersection region common to the
`second microchannel. The flow control regulator of this
`system optionally further regulates flow of the at least one
`fluid in the second and the third microchannels. In this
`embodiment, the computer typically also includes an
`instruction set for simultaneously flowing fluids from the
`second and third microchannels into the first microchannel.
`In particle washing systems, typically, at least fourth and
`fifth channels which intersect the first microchannel at a
`common intersection downstream of the second and third
`microchannels are provided. The computer further includes
`an instruction set for simultaneously flowing material from
`the first microchannel into the fourth and fifth microchan(cid:173)
`nels. Sixth and seventh microchannels which intersect the
`first microchannel at a common intersection downstream of
`the fourth and fifth microchannels, with the computer further
`comprising an instruction set for simultaneously flowing
`40 material from the sixth and seventh microchannels into the
`first microchannel are optionally provided. Similarly, eighth
`and ninth microchannels which intersect the first microchan(cid:173)
`nel at a common intersection downstream of the sixth and
`seventh microchannels, the computer further including an
`45 instruction set for simultaneously flowing material from the
`first microchannel into the eighth and ninth microchannels
`are optionally provided.
`The integrated system optionally includes sources for any
`reagent or particle used in the methods noted above, such as
`50 one or more sources of terminal deoxynucleotide
`transferase, one or more sources of one or more fluorescein
`labeled nucleotides or other labeled polynucleotides, one or
`more sources of Annexin V, one or more sources of an
`AnnexinV-biotin conjugate, one or more sources of a DNA
`55 dye, one or more sources of Campthotecin, one or more
`sources of Calcein-AM, one or more sources of a control
`cell, one or more sources of a test cell, etc.
`Signal detector(s) mounted proximal to the first micro(cid:173)
`channel for detecting a detectable signal produced by one or
`60 more of the members of the at least one particle population
`in the microchannel are typically provided in the integrated
`systems of the invention. The detector also optionally
`includes, e.g., a fluorescent excitation source and a fluores(cid:173)
`cent emission detection element. Optionally, the computer is
`65 operably linked to the signal detector and has an instruction
`set for converting detected signal information into digital
`data.
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`US 6,506,609 Bl
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`6
`Many additional aspects of the invention will be apparent
`upon review, including uses of the devices and systems of
`the invention, methods of manufacture of the devices and
`systems of the invention, kits for practicing the methods of
`