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`PCT/USOI/28667
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`-8-
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`the inlet region. The sample fluid is, specifically, a fluid which is incompatible with the
`extrusion fluid and contains the biological material or sample. Thus, droplets of the
`
`sample fluid containing the biological material for analysis, reaction or sorting are
`
`sheared at the droplet extrusion region into the flow of the extrusion fluid in the main
`channel. Preferably the droplets of the sample fluid each contain, on average, no more
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`than one particle of the biological material. For example, in preferred embodiments
`
`wherein the biological material comprises viral particles, each droplet preferably
`
`contains? on average, no more than one Viral particle. The flow control ofthe device may
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`be adapted to direct the droplets into a branch charmel of the device, «23.53., according to
`
`10
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`a predetermined characteristic of the droplet (or of the. biological material within the
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`droplet} that is detected by a detector as the droplet passes through a detection region of
`
`the detdoe, In preferred embodiments, the extrusion fluid is a. non~polar solvent: such a
`
`decane (8.3. , tetradecane or hexadecane) or another oil (for example? mineral oil}: and the
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`sample fluid is an aqueous solution, such as ultra pure water, a solution of TE- buffer, a
`
`15
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`solution of phosphate buffer saline or a solution of an acetate buffer.
`
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`
`embodiments the extrusion fluid may also contain one or more additives. For example,
`
`in preferred embodiments the extrusion fluid is a non—polar solvent or oil (ego, decane,
`
`tetradecane or hexadeoane} and contains at least one surfactant.
`
`The invention also provides a method for sorting biological material. In various
`
`embodiments ofthe method, the biological material may he, ag. _, molecules (for example,
`
`polynucleotides, polypeptides; enzyunesi substrates or mixtures thereof), cells or viral
`
`particles, or mixtures thereof.
`
`In preferred embodiments, the biological material
`
`comprises Viral particles.
`
`The method: Winch is preferably implemented using a microfehricated detdee of
`
`the invention, comprises steps of: (a) providing droplets ofa sample fluid containing the
`
`biological material to the main channel of a miorofabrieated substrate; (1)) interrogating
`
`each droplet (or the biological material within each droplet) for a predetermined
`
`characteristic as it passes through a detection region associated with the main channel;
`
`and (o) directing the flow of each droplet into a. selected branch channel according to the
`
`30
`
`results of the interrogation. An extrusion fluid, which is incompatible with the sample
`
`fluid, flows through the main channel so that the droplets of the sample fluid are within
`
`the flow of the extrusion fluid in the main channel.
`
`In preferred embodiments, the
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`PCTleStll/2866?
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`Us
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`invention or of any exemplified term. Likewise, the invention is not limited to any
`
`particular preferred embodiments described herein.
`Indeed? many modifications and
`variations of the invention will be apparent to those skilled in the art upon reading this
`
`specification and can be made Without departing horn its spirit and scope. The invention
`
`is therefore to be limited only by the terms of the appended claims along with the full
`
`scope of equivalents to which the claims are entitled.
`
`As used herein? ”about" or ”approximately" shall generally mean within 20
`
`percent, preferably within 19 percent, and more preferably within 5 percent of a given
`value or range.
`I
`
`The term ”molecule" means any distinct or distinguishable structural unit of
`
`matter comprising one or more atoms, and includes for example polypeptides and
`
`polynucleotides.
`
`The term ’jpoiymcr” means any substance or compound that is composed of two
`
`or more building blocks (‘mers") that are repetitively linked to each other. For example,
`
`a "dimer" is a compound in which two building blocks have been joined together.
`
`The term ’incivnucieotide" as used herein refers to a polymeric molecule having
`
`a backbone that supports bases capable ofhydrogen bonding to typical polynucleotides,
`
`where the polymer backbone presents the bases in a manner to permit such hydrogen
`
`bonding in a sequence specific fashion between the polymeric molecule and a typical
`
`polynuoleotide (erg. , single—stranded DNA). Suchbases are typically inosinefl. adenosine,
`
`guanosine, cytosine, uracil and thymidine. Polymeric molecules include double and
`
`single stranded RNA and DNA? and backbone modifications thereofi for example,
`
`methylphosphonate linkages.
`
`Thus, a "polynucleotide" or "nucleotide sequence" is a series ofnucleotide bases
`
`(also called "nucleotides") generally in DNA and RNA: and means any chain of two or
`
`more nucleotides. Anttcleotide sequence typically carries genetic information, including
`
`the information used by cellular machinery to make proteins and enzymes. These terms
`
`include double or single stranded genomic and cDNA, RNA, any sgmthetic and
`
`genetically manipulated polynucleotida and both sense and anti—sense polynucleotide
`
`(although only sense smnds are being represented herein). This includes single- and
`
`double—stranded molecules, i. a. _. DNA—DNA, DNA-MA and Elba-RNA hybrids, as well
`
`as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid
`
`

`

`‘WO (12/253163
`
`PCTleSfll/lfidfi?
`
`-14-
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`backbone. This also includes nucleic acids containing modified bases, for example thic-
`
`uracil, thin—guanine and fluoro-uracil.
`
`The pen-nucleotides herein may be flanked by natural regulatory sequences, or
`
`may be associated with heterologous sequences,
`
`including promoters, enhancers,
`
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`response elements, signal sequences, polyadenylation sequences, introns, 5'» and 3'- non-
`
`coding regions, and the like. The nucleic acids may also be modified by many means
`
`known in the art. Nonelimiting examples of such modifications include methylation,
`
`"caps", substitution ofone or more ofthe naturally occurring nucleotides with an analog,
`
`and internucleotide modifications such as, for example, those with 1mcharged linkages
`(e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and
`
`10
`
`with charged linkages
`
`(e.g., phosphorothicates,
`
`phosphorodithioates,
`
`etc).
`
`Polynueleotides may contain one or more additional covalently linked moieties, such as,
`
`for example, proteins (e. g. , nucleases, toxins, antibodies, signal peptides, poly-L—lysine,
`
`etc), intercalators (cg. , acridine, psoralen, etc), chelators (cg. , metals, radioactive
`
`metals, iron, oxidative metals, etc), and allcylators. The polynucleolides may be
`
`. derivatized by formation of a methyl or ethyl phosphotriester or an allqd
`
`phosphoramidale linkage. Furthermore, the poly-"nucleotides herein may also be modified
`
`with a label capable of providing a detectable signal, either directly or indirectly.
`
`Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
`
`’fDAid ” (deoayribonucleio acid) means any chain or sequence of the chemical
`
`building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide
`
`bases, that are linked together on a deoxydibose sugar backbone. DNA can have one
`
`strand ofnucleotide bases, or two complimentary strands which may form a double helix
`
`structure.
`
`“SEMI” (ribonucleic acid) means any chain or sequence of the chemical
`
`building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide
`
`bases, that are linked together on aribose sugar backbone. RNA typically has one strand
`
`of nucleotide bases.
`
`A ’jpolflacpz'z'de” (one or more peptides) is a chain of chemical building blocks
`
`called amino acids that are linked together by chemical bonds called peptide bonds. A
`
`’jnrorein " is a polypeptide produced by a livmg organism. A protein or polypeptide may
`
`be "native" or TWild-type”, meaning that it occurs in nature; or it may be a "mutan ",
`
`"varian ," or ”modified", meaning that it has been made, altered, derived, or is in some
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`PCT/USit1/28667
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`10
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`as a marker. The marker may be directly or indirectly associated with the reporter or can
`
`itself be a reporter. Thus. a marker is generall}.r a distinguishing feature of a molecule.
`
`cell or virion, and a reporter is generally an agent which directly or indirectly identifies
`
`or permits measurement of a marker,
`
`These terms may: however. be used
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`interchangeably.
`
`The term ’flow " means any movement of liquid or solid through a device or in
`
`a method ofthe invention, and encompasses without limitation any fluid stream, and any
`
`material moving with, within or against the stream, whether or not the material is carried
`
`by the stream. For example, the movement of molecules, cells or virions through a
`
`device or in a method. ofthe invention, cg. through channels ofa microtluidic chip ofthe
`
`invention, comprises a flow. This is so, according to the mvenlion: whether or not the
`
`molecules, cells or Visions are carried by a stream of fluid also comprising a flow, or
`
`whether the molecules; cells or virions are caused to move by some other direct or
`
`indirect force or motivation, and whether or not the nature of an}r motivating force is
`
`known or understood. The application of any force may he used to profide a flow,
`
`including without limitation, pressure, capillary action, electric-osmosis, electrophoresis,
`
`dielectrophoresis, optical tweezers, and combinations thereof, without regard for any
`
`particular theory or mechanism of action, so long as molecules. cells or Virions are
`
`directed for dctectiom measurement or sorting according to the invention.
`
`An ”inlet region "' is an area of a microfahricated chip that receives molecules,
`
`cells or virions for detection measurement or sorting. The inlet region ma},r contain an
`
`inlet charmel. a well or reservoir, an opening, and other features which facilitate the entry
`
`of molecules, cells or Virions into the device. A chip may contain more than one inlet
`
`region if desired. The inlet region is in fluid connnmlication with the main channel and
`
`is upstream therefrom.
`
`An ”carrier region ” is an area of a microfahricated chip that collects or dispenses
`
`molecules, cells or unions afier detection, measurement or sorting. An outlet region is
`
`downstream from a discrimination region and may contain branch channels or outlet
`
`channels. A chip near}r contain more than one outlet region it'desired,
`
`An "’orzalysis unit" is a microfahricated substrate, tag. a microfiibrieated chip,
`
`having at least one inlet region, at least one main channel. at least one detection region
`
`and at least one outlet region. Sorting embodiments of the analysis unit include a
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`charms]. A reservoir may facilitate introduction ofmolecules or cells into the device and
`
`into the sample inlet channel ofeach analysis unit. An inlet region may have an opening
`
`such as in the floor of the microfabricated chip, to permit entry of the sample into the
`
`device. The inlet region. may also contain a connector adapted to receive a suitable piece
`
`of tubing, such as liquid cinematography or I-IPLC tubing, through which a sample may
`
`be supplied. Such an arrangement facilitates introducing the sample solution under
`
`positive pressure in order to achieve a desired pressure at the droplet extrusion region.
`
`A. device of the invention may have an additional inlet region,
`
`in direct
`
`communication with the main charms] at a location upstream of the droplet extrusion
`
`region, through which a pressurized stream or "HOW" of a fluid is introduced into the
`
`main channel. Preferably, this fluid is one which is not miscible with the solvent or fluid
`
`of the sample. For example, most preferably the fluid is a non—polar solvent, such as
`
`decane tags, tetradecane or hexadecane), and the sample (sag, of cells, virions or
`
`molecules) is dissolved. or suspended in an aqueous solution so that aqueous droplets of
`
`the sample are introduced into the pressurized stream of non-polar solvent at the droplet
`
`extrusion region,
`
`Substrate and Flow Channels
`
`A tgpical analysis unit ofthe invention comprises a main inlet that is part of and
`
`feeds or communicates directljgr with a main channel, along with one or more sample ~
`
`inlets in communication with the main channel at a droplet extrusion region situated
`
`downstream from the main inlet (each different sample inlet preferably corrununicates
`
`with the main channel at a different droplet extrusion region). The droplet extrusion
`
`region, generally comprises a junction between the sample inlet and the main charmel
`
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`
`such that a pressurized solution of a sample (116., a fluid containing a sample such as
`
`cells, virions or molecules) is intmduced to the main chaimel in droplets. Preferably, the
`
`sample inlet intersects the main channel such that the pressurized sample solution is
`
`introduced into the main channel at an angle perpendicular to a stream of fluid passing
`
`through the main channel. For example, in preferred embodiments, the sample inlet and
`
`main channel intercept at a T—shaped junction;
`
`ta, such that the sample inlet is
`
`perpendicular (:90 degrees} to the main channel. However, the sample inlet may intercept
`
`the main channel at any angle, and need not introduce the sample fluid to the main
`
`

`

`WO 02/23163
`
`PCT! USN/286157
`
`.25-
`
`channel at an angle that is perpendicular to that flow. In exemplary embodiments the
`
`angle between intersecting channels is in the range of finm about 60 to about 120
`
`degrees. Particular enzemplarg,r angles are 45? 60, 90, and 120 degrees.
`
`The main channel in turn communicates with two or more branch channels at
`
`another junction or "branch point".. forming, for example, a T—shape or a szhape. (Ether
`
`shapes and channel geometries may he used as desired. In sorting embodiments, the
`
`region at or sunntmding the jiniction can also be referred to as a discrimination region.
`
`Precise boundaries for the discrimination region are not required, but are preferred.
`
`A detection region is within, conmnmieating or coincident with a portion of the
`
`10
`
`main charmel at or downstream of the droplet extrusion region and,
`
`in sorting
`
`embodiments, at or upstream of the discrimination region or branch point. Precise
`
`boundaries ' for the detection region are not required. but are preferred.
`
`The
`
`discrimination region may be located innnediately downstream of the detection region
`
`or it may be separated by a suitable distance consistent with the size ot‘the molecules, the
`
`channel dimensions and the detention system. It will be appreeiated that the channels
`
`may have any suitable shape or crossxsection (for example, tubular or greeted), and can
`
`be arranged in any suitable manner so long as flow can be directed from inlet to outlet
`
`and item one channel into another.
`
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`The channels of the invention are microfabricated, for example by etching a
`
`silicon chip using conventional photolithography techniques: or using a mic-tom achining
`
`teohrmlog3.r called "soft lithography", developed in the late 1990's (19). These and other
`
`microfabrication methods mayr be used to provide inexpensive miniaturized devices, and
`
`in the ease of soft lithography, can provide robust devices having beneficial properties
`
`such as improved flexibilit}; stability, and mechanical strength. When optical detention
`
`is employed, the invention also provides minimal light scatter from molecule or cell
`
`{including Virion) suspension and chamber material. Devices according to the invention
`
`are relatively inexpensive and easy to set up. They can also be disposable; which greatly
`
`relieves many ofthe concerns of gel electrophoresis (for molecules), and of sterilization
`
`and pennanent adsorption of particles into the flow chambers and charmels of
`
`conventional FAQS machines (for cells, virions and other particle suspensions). Using
`
`these kinds of techniques. microfabricated fluidie devices can replace the conventional
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`fluidic flow chambers of the prior art.
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`microns, preferably about 60 microns, and more preferably about 30 microns at the
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`erossflow area or droplet extrusion region. This geometry facilitates an orderly flow of
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`droplets in the channels, See eg. FIG. 163. Similarly, the volume ofthe detection region
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`in an analysis device is typically in the range of between about 10 ferntoliters (fl) and
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`5000 H, preferably about 40 or 50 fl to about 1000 or 2000 ii, most preferably on the
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`order of about 200 f1.
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`In preferred embodiments, the charmels of the device, and
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`particularly the ehannels ofthe inlet connecting to a droplet extrusion region, are between
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`about 2 and 50 microns, most preferably about 30 microns.
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`in one preferred embodiment, droplets at these dimensions tend. to conform to the
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`droplets more from a wider channel to a narrower channel they become longer and
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`
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`lozenge shape, flows smoothly and well through the channels. Longer droplets, produced
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`in narrower channels, provides a higher shear, meaning that droplets can more easily be
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`sheared or broken offfi'orn a flow, is. using less force Droplets may also tend to adhere
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`to channel surfaces, which can slow or block the flow, or produce turbulence, Droplet
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`adherence is overcome when the droplet is massive enough in relation to the channel size
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`to break free. Thus, droplets of varying size, if present, may combine to form rmiform
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`droplets having a so~ealled critical mass or volume that results in smooth or laminar
`
`droplet flow. Droplets that are longer than they are wide, preferably about four times
`longer than they are Wide, generally have the ability to overcome channel adherence and
`
`move freely through the mierofluidic device. Thus, in an exemplary embodiment with
`
`60 micron channels, atypical flee-flowing droplet is about 60 microns Wide and 240
`
`microns long. Droplet dimensions and flow characteristics can be influenced as desired,
`
`in part by changing the channel dimensions, 8.3. the channel width.
`
`More preferably, however, the microfahrieated devices ofthis invention generate
`
`round, monodisperse droplets t:such as those illustrated in Frames J and L of FIG. 19).
`
`Preferably, the droplets have a diameter that is smaller than the diameter of the
`
`microeharmel;
`
`tie, preferably less than 60 um. Monodisperse droplets may be
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`particularly preferably, 6.3., in high throughput defines and other embodiments where
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`it is desirable to generate droplets at high frequeney.
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`‘WO 02/231d3
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`PCTi’USOl/ZSGG?
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`-34-
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`Where a lamp is used, the chamiels are preferably shielded fi‘om 1ight in all regions except
`
`the detection region. In embodiments where a laser is used, the laser can be set to scan
`
`across a set of detection regions from different analysis units. In addition, laser diodes
`
`may he Iliicrofabricated into the same chip that. contains the analysis units. Alternatively,
`
`laser diodes may be incorporated into a. second chip (2‘. e. , a laser diode chip) that is placed
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`region. This design provides the advantages of compactness and a. shorter optical. path
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`for exciting andj'or emitted radiation, thus minimizing distortion.
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`Sorting Schemes
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`According to the invention, molecules (such as DNA, protein, enzyme or
`
`substrate) or particles (is. , cells, including, virions) are sorted dynamically in a flow
`
`stream of microscopic dimensions based on the detection or measurement of a
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`characteristic, marker or reporter that is associated with the molecules or particles. More
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`specifically, droplets ofa solution (preferably an aqueous solution or buffer), containhig
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`a sample cfmolecules, cells or videos, are introduced through a droplet extrusion region
`
`into a stream offluid (preferably a nonnpolar fluid such as decane or other oil) in the main
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`channel. The individual droplets are then analyzed andlor sorted in the flow stream,
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`thereby sorting the molecules, cells or Virions contained within the droplets.
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`The flow stream in the main channel is typically, but not necessarily continuous
`
`and may he stopped and started, reversed or changed in speed. Prior to sorting, a liquid
`
`mLn
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`that does not contain samples molecules, cells or virions can he introduced into a sample
`
`inlet region (such as an inlet well or channel) and directed through the droplet extrusion
`
`region, eg, by capillarj,r action, to hydrate and pnepare the device for use. Likewise,
`
`buffer or oil can also be introduced into a main inlet region that communicates directly
`
`with the main channel to purge the device fag, or "dead" air) and prepare it for use. If
`
`desired, the pressure can be adjusted or equalized, for example, by adding buffer or oil
`
`to an outlet region.
`
`The pressure at the droplet extrusion region can also be regulated by adjusting the
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`W0 02/353163
`
`PCT/U SIJIIZ‘SGé?’
`
`-35-
`
`pressure on the main and sample inlets, for example, with pressurized syringes feeding
`
`into those inlets. By controlling the pressure difference between the oil and water
`
`sources at the droplet extrusion region, the size and periodicity of the droplets generated
`
`may be regulated. Alternatively, a valve may he placed at or coincident to either the
`
`droplet esttusion region or the sample inlet connected thereto to control the flow of
`
`solution into the droplet extrusion region, thereby controlling the size and periodicity of
`the droplets. Periodicity and droplet volume may also depend on channel dienreten the
`
`viscosity of the fluids? and sheer pressure.
`
`The droplet forming liquid is typicelljlr an aqueous buffer solution; such as
`
`10
`
`ultrapure water (rag, 18 mega-ohm resistivity, obtained, for example by column -
`
`chromatography)? 10 113M Tris HCl and 1 mM EDTA (TE) huf' er, phosphate buffer
`
`saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible
`
`with the population ofr110leculesE cells or Vii-ions to be analyzed andj'or sorted can he
`
`used The fluid passing through. the main channel and in which the droplets are formed
`
`is preferably one that is not miscible with the droplet forming fluid. Preferably: the fluid
`
`passing through the main channel is a non—polar solvent, most preferably decent-1 (eg,
`
`tetradecane or hexadecane) or another oil.
`
`The fluids used in the invention may contain additives, such as agents which
`
`reduce surface tensions (surfactants). Exemplary surfactants include Tweenl Span:
`
`fluorinated oils, and other agents that are soluble in oil relative to water. Surfactmlts may
`
`aid in controlling or optimizing droplet size, floatr and uniformity, for example by
`
`reducing the shear force needed to extrude or inject droplets into an intersecting channel.
`
`This may affect droplet volume and periodicity= or the rate or frequency at which droplets
`
`break off into an intersecting channel.
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`[\J (J:
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`Channels of the invention may he formed from silicon elastomer (eg. RTV),
`
`urethane compositions, offi‘om silicon-urethane composites such as those available Erorn
`
`Polymer Technology Group (Berkeley, CA), cg; lE‘urSilfl"i and CarboSilTM, The channels
`
`may also be coated with additives or agents, such as surfactants, TEFLQN, or fluoiineted
`
`oils such as octadecafluoroctene (98%, Aldrich) or fl‘uorononane. TEFLON is
`
`particularly suitable for silicon elastomer (RTE/l) charmels. which are hydrophobic- and
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`advantageously do not absorb water, but they may tend to swell when exposed. to en oil
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`phase. Swelling may alter chemiel dimensions and shape, and may even close off
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`'WO 021'33163
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`PCTIUSOIJZ-‘jfié?
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`will contain two or more molecules, cells or vii-ions. This is to ensure that for the large
`
`majority of measurements, the level of reporter measured in each droplet as it passes
`
`through the detection region corresponds to a single nioleoule, cell or virion and not to
`
`two or more molecules, cells or virions.
`
`The parameters which govern this relationship are the volume ofthe droplets and
`
`the concentration of molecules, cells or virions in the sample solution. The probability
`
`that a droplet will contain two or more molecules cells or Virions {Pa} can be expressed
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`as
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`S 2 = 1" {1+ [Virion] s V} x e
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`where "[t-irion]fl is the concentration of molecules, cells or Vitions in units of number of
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`units of unia.
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`
`of molecules, cells or virions in the sample solution. However, decreasing the
`
`concentration of molecules, cells or Virions in the sample solution also results in an
`
`increased volume of solution processed through the device and can result in longer run
`
`times. Accordingly, it is desirable to minimize to presence of multiple molecules, cells
`
`or Virions in the droplets (thereby increasing the accuracy of the sorting) and to reduce
`
`the volume of sample, thereby pennitting a sorted sample in a reasonable time in a
`
`reasonable volume containing an acceptable concentration ofmolecules, cells or Virions.
`
`The maximum tolerable P52 depends on the desired "purity" ofthe sorted sample.
`
`The "purity" in this case refers to the fraction of sorted molecules, cells or virions that
`
`posses a desired characteristic (eg, display a particular antigen, are in a specified size
`
`range or are a partieular type ofmolecule, cell or vii-ion). The purity ofthe sorted sample
`
`is inversely proportional to Pig. For example, in applications where high purity is not
`
`needed or desired a relatively high P5,, (age, Page 0.2.) may be acceptable. For most
`
`applications, maintaining P,2 at or below about 0.1, preferably at or below about 0.01,
`
`provides satisfactory results.
`
`A sample solution containing amixture or population ofmolecule, sells or VlI‘lQI‘lS‘
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`in a suitable carrier fluid (such as a liquid or butter described above) is supplied to the
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`sample inlet region, and droplets of the sample solution are introduced, at the droplet
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