WSGRDocket No. 38938-743.101
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`PROVISIONAL PATENT APPLICATION
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`COMPOSITIONS AND METHODS FOR DROPLET FORMATION, SPACING AND
`
`DETECTION
`
`Inventors:
`
`Erin Steenblock
`Citizen of the United States of America, Residing at
`1000 Alfred Nobel Drive
`Hercules, CA 94547
`
`Amy Hiddessen
`Citizen of the United States of America, Residing at
`5050 Hacienda Drive
`Dublin, CA 94568
`
`Ben Hindson
`Citizen of the United States of America, Residing at
`1039 Bannock St.
`Livermore, CA 94551
`
`Kevin Ness
`Citizen of Canada, Residing at
`4894 Bernal Avenue, #J
`Pleasanton, CA 94566
`
`Assignee:
`
`Bio-Rad Laboratories, Inc.
`1000 Alfred Nobel Drive
`Hercules, CA 94547
`
`A Delaware corporation
`ur —_ -WR
`Wilson Sonsini Goodrich & Rosati
`PROPS SSTONAL CORPORATION
`
`650 Page Mill Road
`
`Palo Alto, CA 94304
`
`(650) 493-9300 (Main)
`
`(650) 493-6811 (Facsimile)
`
`Filed Electronically on: September 7, 2012
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`COMPOSITIONS AND METHODS FOR DROPLET FORMATION, SPACING, AND
`
`DETECTION
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`BACKGROUNDOF THE INVENTION
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`[0001] Assays for determining the presence, quantity, activity, and/or other properties or
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`characteristics of components in a sample play a valuable role in many diverse biological and
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`clinical applications. In somecases, the componentsof interest within a sample—e.g., a nucleic
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`acid, an enzyme, a virus, a bactertum—are only minorconstituents of the sample and may,
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`therefore, be difficult to detect or quantitate.
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`[0002] Certain biological assays, such as the polymerase chain reaction (PCR) assay, can be
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`quantitative in specific settings. For example, real-time PCR (which generally involves
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`monitoring the progression of amplification using fluorescence probes) can permit quantification
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`of target nucleic acids in a sample, particularly where the target nucleic acids are somewhat
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`abundant.
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`[0003] Digital PCRis also a quantitative PCR assay. In digital PCR, a sample containing PCR
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`reagents and target nucleic acid moleculesis distributed across multiple partitions, often such
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`that each individual partition contains on average one or fewertarget nucleic acid molecules.
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`After amplification, reactions containing one or more templates are generally detectable and can
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`emit a signal such as a fluorescent signal. Droplet digital PCR is a form of digital PCR that uses
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`fluidic droplets for the partitions. The steps for droplet digital PCR generally involve (1)
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`partitioning a fluid sample containing PCRreagents and nucleic acid target molecule(s) into
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`multiple droplets, (2) performing an amplification cycle on the droplets, and (3) detecting the
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`presence of nucleic acids in the droplets. A nucleic acid sample can be partitioned into multiple
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`droplets using oil and emulsion chemistry. For example, an aqueous sample can be partitioned
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`into multiple emulsified droplets in a continuousoil phase using microfluidics technologies.
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`SUMMARYOF THE INVENTION
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`[0004] In one aspect, the present disclosure provides a system, device or kit for detecting
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`droplets, comprising: (a) a detector device comprising an input flow path, an intersection region,
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`and an output flow path, wherein the intersection region is downstream ofthe input flow path
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`and the output flow path is downstream ofthe intersection region; (b) droplets located within the
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`input flow path; and (c) an aqueousfluid for separating the droplets wherein the droplets are
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`introduced to the aqueousfluid at the intersection region. The input flow path may comprise a
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`continuous phase of non-aqueousfluid. In some embodiments, the non-aqueousfluid is an
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`aqueous-immiscible fluid. In a further embodiment, the non-aqueousfluid is an oil. The output
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`flow path may comprise a continuous phase of aqueousfluid. In some embodiments, the
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`aqueousfluid comprises a surfactant. The droplets in the output flow path may have an inner
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`core containing an aqueousfluid that is encapsulated with a non-aqueousfluid. In some
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`embodiments, the non-aqueousfluid is a continuous phase. In some other embodiments, the
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`non-aqueousfluid is a discontinuousphase. Alternatively, the output flow path may comprise a
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`continuous phase of non-aqueousfluid. In some embodiments, the inner wall of the output flow
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`path is covered by the aqueousfluid. In some embodiments, emulsified droplets flow out of the
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`output flow path in a stream which hasa diameter substantially smaller than the diameter of the
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`output flow path. In another aspect, the present disclosure provides a system for detecting
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`droplets, comprising: (a) a detector device comprising an input flow path, an intersection region,
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`and an output flow path, wherein the intersection region is downstream ofthe input flow path
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`and said output flow path is downstream ofsaid intersection region; and (b)
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`an oil-
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`immiscible fluid for separating said droplets, wherein said oil-immiscible fluid is introduced to
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`said droplets at said intersection region. In some case, the continuousphase offluid within the
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`input flow path is a non-aqueousfluid and the inner surface of the output flow path is coated
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`with the oil-immiscible fluid.
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`[0005] Additionally, the present disclosure provides methods for separating droplets. In one
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`aspect, the present disclosure provides a method of separating droplets, comprising: (a) flowing a
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`stream of non-aqueousfluid comprising said droplets along a flow path comprising: (i) an input
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`flow path,(ii) an intersection region, and (iii) a downstream output flow path; and (b)
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`introducing a stream ofoil-immiscible fluid to said intersection region; wherein the average
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`distance between said droplets in said output flow path is greater than the average distance
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`between said droplets within said input flow path. In another aspect, the present disclosure
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`provides a method of separating droplets, comprising: (a) flowing a stream of non-aqueousfluid
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`comprising the droplets along a flow path comprising: (i) an intersection region and(11) a
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`downstream output flow path; and (b) introducing a stream ofoil-immiscible fluid to said
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`intersection region; wherein said droplets are heated prior to entering said intersection region. In
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`yet another aspect, the present disclosure provides a methodof detecting droplets, comprising:
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`(a) flowing a stream of non-aqueousfluid through a continuous flow path comprising an
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`intersection region and a downstream detection region, wherein said non-aqueous fluid
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`comprises said droplets; (b) introducing a stream ofoil-immiscible fluid to said intersection
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`region; and (c) detecting a signal from the droplets as they pass through said downstream
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`detection region. The output flow path may comprise: (a) a continuous phaseofoil-immiscible
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`fluid; and (b) aqueous droplets encapsulated by a layer of non-aqueousfluid. Additionally, the
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`flow paths of the non-aqueousfluid and that of the oil-immiscible fluid may have different
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`angles, ranging from 1 degree to 90 degree inclusive. In one embodiment, the two flow paths are
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`substantially perpendicular.
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`[0006] The oil-immiscible fluid may comprises air. Alternatively, the oil-immiscible fluid may
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`comprise water. When the oil-immiscible fluid comprises water, the water may compriseat least
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`one additive. Theat least one additive may adjust properties of water, for example, surface
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`tension, viscosity, tendency to foam andanti-bacteria or anti-microbial activity. Example of
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`additions may include, but are not limited to, surfactant, glycerol, antimicrobial agent and
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`antifoaming agent. Any of these above mentioned agents can be uses alone or in combination. In
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`somecase, the oil-immiscible fluid comprisesat least one surfactant and glycerol. In some other
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`cases, the oil-immiscible fluid comprises at least one surfactant, at least one antimicrobial agent
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`and glycerol.
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`[0007] The surfactant may be ionic or non-ionic. In somecases, the surfactant is a block
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`copolymerof polypropylene oxide and polyethylene oxide. In some cases, the surfactantis a
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`fluorinated surfactant. The fluorinated surfactant may be negatively charged or may comprise a
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`carboxylate group. The amountof surfactant used may depend on the desired properties of the
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`fluid. The weight of the surfactant may beat least 0.001%, at least 0.01%, at least 0.1%, at least
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`1%, at least 5% or even more of the weight of the fluid they are added to. In some cases, the
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`amount of surfactant is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
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`7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%
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`or about 20%. In some cases, the amount of surfactant is in a range of 0.1%-99% about 1%-
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`99%, 3%-99%, 4%-99%, 5%-99%, 10%-99%, 1%-20%, 1%-30% or 1%-40 the weight of the
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`fluid they are addedto.
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`[0008] The non-aqueousfluid may comprise an oil selected from the group consisting of a
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`silicone oil, a mineraloil, a hydrocarbonoil, a fluorocarbon oil, a vegetable and a soybeanoil. In
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`some embodiments, the non-aqueousfluid comprise a surfactant. The droplets may be aqueous
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`droplets encapsulated by the non-aqueousfluid. Upon flowingto the intersection region, the
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`droplets may be further emulsified. The flowing can be achieved with under negative or positive
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`fluidic pressure. In some embodiments, the flowing is achieved with at least one syringe pump.
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`[0009] The present disclosure enables detection of droplets with different sizes and properties. In
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`some cases, the droplets have varying sizes. In some cases, the droplets are emulsified droplets.
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`The droplets may comprise a nucleic acid or a product of a nucleic acid amplification reaction. In
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`a further embodiments, each of the droplets, on average, comprisesless than five target nucleic
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`acids.
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`[0010] The use of a non-aqueousfluid and an oil-immiscible fluid may create a virtual capillary
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`in the output flow path. The inner wall of the output flow path may be coated withthe oil-
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`immiscible fluid, thus reducing aperture of the output flow path. The thickness of the coating
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`layer may beatleast 0.01%,at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at
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`least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at
`
`least 70%, at least 80%, at least 90% or even moreof the diameter of the output flow path. In
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`some cases, the thickness maybe in a range of 1%-90%, 5%-90%, 10%-90%, 15%-90%, 20%-
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`90%, 25%-90%, 30%-90%, 40%-90%, 50%-90%, 5%-95%, 10%-95%, 15%-95%, 30%-95% or
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`50%-95% of the diameter of the output flow path. The formation ofa virtual capillary may
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`allow the droplets flowing through the output flow path serially and substantially centered,
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`regardless oftheir sizes.
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`[0011] Additional aspects and advantagesof the present disclosure will becomereadily apparent
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`to those skilled in this art from the following detailed description, wherein only illustrative
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`embodiments of the present disclosure are shown and described. As will be realized, the present
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`disclosure is capable of other and different embodiments, andits several details are capable of
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`modifications in various obviousrespects, all without departing from the disclosure.
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`Accordingly, the drawings and description are to be regardedasillustrative in nature, and not as
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`restrictive.
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`INCORPORATION BY REFERENCE
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`[0012] All publications, patents, and patent applications mentionedin this specification are
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`herein incorporated by reference in their entireties to the same extent as if each individual
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`publication, patent, or patent application was specifically and individually indicated to be
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`incorporated by reference.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0013] The novel features of the invention are set forth with particularity in the appendedclaims.
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`A better understanding of the features and advantagesof the present invention will be obtained
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`by reference to the following detailed description that sets forth illustrative embodiments, in
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`whichthe principles of the invention are utilized, and the accompanying drawings of which:
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`[0014] FIG.1 illustrates a general workflow for droplet digital PCR (ddPCR) technology.
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`[0015] FIG.2 illustrates an exemplary flowchart depicting the steps of a fluorescence detection
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`method in a flow-based system.
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`[0016] FIG.3 illustrates an exemplary device for spacing and detecting droplets in a flow
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`system.
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`[0017] FIG.4 illustrates another exemplary device for spacing and detecting droplets in a flow
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`system.
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`[0018] FIG. 5 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water.
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`[0019] FIG.6 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water and 8% glycerol.
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`[0020] FIG.7 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water and 16% glycerol.
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`[0021] FIG.8 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water and 1% Pluronic®
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`surfactant (upper panel) or with an oil-immiscible fluid comprising water, 8% glycerol, and 2%
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`Pluronic® surfactant (lower panel).
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`[0022] FIG.9 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water (upper panel) or
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`with a focusing fluid comprising an oil (lower panel).
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`[0023] FIG. 10 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are flowed through a detector device using a 10:1 singulationratio.
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`[0024] FIG. 11 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with an oil-immiscible fluid comprising water, 8% glycerol, and
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`2% Pluronic® F-68 surfactant.
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`[0025] FIG.12 is a graphical representation of the fluorescence amplitudes of droplets detected
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`after the droplets are contacted with a focusing fluid comprising HFE-7500oil.
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`[0026] FIG. 13 is a graphical representation of detected signal after the droplets are contacted
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`with a focusing fluid and either the tip is not wiped(left panel) or the tip is wiped (right panel).
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`DETAILED DESCRIPTION OF THE INVENTION
`
`Overview
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`[0027] The present disclosure provides methods, devices, compositions, kits, and systems for
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`separating and detecting emulsified droplets, generally within a detector device. Often, the
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`detector device comprises an input flow path (e.g., channel, tube, capillary, etc.) connected to at
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`least one intersection region that is connected to an output flow path. The droplets may flow
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`through the input flow path within a particular fluid; and then, at or near the intersection region,
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`a fluid that is immiscible with that particular fluid may be introduced to the droplet emulsion.
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`The immiscible fluid may be delivered throughat least one delivery flow path to the intersection
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`region. The emulsified droplets in the output flow path generally flow to at least one
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`downstream detection region. In somecases, the detector device comprises a detector that
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`detects a signal emitted from the emulsified droplets; such detection may occurin a detection
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`region.
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`[0028] The methods and devices provided herein may enable modulation of the spacing between
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`droplets. For example, the device may increase the spacing between droplets in the output flow
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`path. This increase in spacing mayoccurasa result of the introduction of an immiscible fluid at
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`the intersection region. In somecases, the average distance between the droplets in the output
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`flow path maybe greater than that between the droplets in the input flow path. In some cases,
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`the device may be able to decrease, or otherwise modulate, the spacing between the droplets.
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`[0029] The fluids used in the devices described herein may be oil-immiscible (e.g., aqueous, air,
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`etc.) or non-aqueous, or a combination of both. In some embodiments, the non-aqueousfluid is
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`an oil. The oil may be selected from the group consisting of a silicone oil, a mineraloil, a
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`hydrocarbon,a fluorocarbon oil, a vegetable and a soybean oil. The aqueousfluid may be any
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`appropriate aqueousfluid including water.
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`[0030] The immiscible fluid that is introduced to the droplets at or near the intersection may
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`form a streaming layer along the interior surface of the output flow path, thereby forminga track
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`or virtual capillary. (See, e.g., 308 of Figure 3.). Such immiscible fluid can be any fluid
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`immiscible with the continuous phaseofthe fluid in the input flow path. The fluid may be
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`aqueous or non-aqueous,air or liquid, etc. For example, the input flow of fluid may comprise
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`aqueousdroplets flowing in a continuous phase comprising oil (or other non-aqueousfluid). An
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`oil-immiscible fluid (e.g., aqueous, water, air) may then be introduced suchthat the aqueous
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`droplets may then travel along the oil-immiscible fluid virtual capillary layer or track as the
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`droplets flow through the output flow path.
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`[0031] The virtual capillary may alter the aperture of the output flow path. The alteration may
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`be achieved by coating the inner wall of the output flow path with the oil-immiscible fluid, or
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`other fluid. The thickness and/or the size of cross-section of this track or virtual capillary may be
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`adjusted in order to accomplish focusing of the droplets, or positioning of the droplets along a
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`particular dimension(s). In somecase, the thickness and/or the size of cross-section ofthis track
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`or virtual capillary is adjusted by adjusting the viscosity and/or surface tension ofthe oil-
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`immiscible fluid.
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`[0032] In somecases, this disclosure provides droplet-size independent methods of separating
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`and detecting droplets. For example, the virtual capillary may enable detection of droplets,
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`irrespective of the size of the droplets. In a further embodiment, droplets contained in the virtual
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`capillary are similar in size to the droplets in the input flow channel. In somecases, the virtual
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`capillary may enable detection of a population of droplets of different sizes (such as
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`polydispersed droplets) and/or of different shapes. In somecases, the droplets flow along the
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`virtual capillary to a detection region and are detected. In someother cases, the droplets flow
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`along the virtual capillary in a single file and substantially centered, independentoftheir sizes.
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`[0033] In another aspect, the droplets may be formed as multiple emulsions(e.g., double
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`emulsions, triple emulsions, quadruple emulsions, etc.). In some cases, double emulsified
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`droplets are formed with diameters about the diameter of the output flow channel are formed; in
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`other cases, the double-emulsified droplets have diameters that are much shorter than the
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`diameter of the output flow channel.
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`[0034] The droplets described in this disclosure are useful in many applications. Often, they
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`contain target nucleic acid(s) and/or materials necessary to carry out an amplification reaction of
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`the target nucleic acid (e.g., polymerase chain reaction (PCR)). In somecases, the droplets may
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`be heated, or subjected to thermal cycling. This can occurprior to, during, or after droplet
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`separation (e.g. prior to entering the input flow path and/orprior to reaching an intersection
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`region). In many cases, PCR is performed in the droplets; in other cases, a reaction other than a
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`PCRreaction occurs within the droplets.
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`[0035] As used herein the term “‘about” a certain value encompassesexact value as well as
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`
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`values within +10 % of such value, and includes values within the range of 0 to +10%,including
`
`
`
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`+1, 2,3, 4, 5, 6, 7, 8, 9, and 10% as well as values less than +1%, such as +0.1, .2, .3, .4, .5, .6,
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`.7, .8, or 9%.
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`Workflows
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`[0036] FIG. 1 depicts a workflow for droplet digital PCR (ddPCR)technology. In brief, the
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`workflow may include a sample preparation step 100, followed by a droplet generation step 102,
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`a reaction step 104 (e.g., amplification, PCR,etc.), a detection step 106, and a data analysis step
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`108. The sample preparation step 100 may involve collecting a sample, such as a clinical or
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`environmental sample, and treating the sample to release associated nucleic acids for PCR
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`amplification. The droplet generation step 102 may involve partitioning the nucleic acids into
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`multiple droplets. In addition to the target nucleic acid for amplification and detection, other
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`reagents, such as a DNA polymerase(e.g., a heat-stable DNA polymerase, such as Taq
`polymerase), a heat-stable ligase, a dNTPs, magnesium (e.g., Mg”), a primerfor a nucleic acid
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`target, among others, may be included. Droplet generation can also involve encapsulating dyes,
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`such as fluorescent molecules, in droplets, for example, with a known concentration of dyes,
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`where the droplets are suspended in an immiscible carrier fluid, such as oil, to form an emulsion.
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`The reaction step 104 may involve subjecting the droplets to a suitable reaction, such as thermal
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`cycling to induce PCR amplification, so that target nucleic acids, if any, within the droplets are
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`amplified to produce additional copies. PCR may be performed by thermal cycling between two
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`or more temperature set points, such as a higher melting (denaturation) temperature and a lower
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`annealing/extension temperature, or among three or more temperature set points, such as a higher
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`melting temperature, a lower annealing temperature, and an intermediate extension temperature,
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`amongothers. A detection step 106 may involve detecting somesignal(s) from the droplets, as
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`an indication as to whether or not there was amplification. Finally, a data analysis step 108 may
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`involve estimating the quantity of target nucleic acid in a sample based on the percentage of
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`droplets in which amplification occurred.
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`[0037] FIG.2 is a flowchart generally depicting steps of a method of detecting or reading
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`droplets. Although various steps of method 200 are described below and depicted in FIG. 2, the
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`steps need not necessarily all be performed, and in some cases may be performedin a different
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`order than the order shown in FIG. 2. Droplets containing a sample (e.g., nucleic acids) may be
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`loaded into an input flow path 202. The droplets may have been heated or subjected to thermal
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`cycling before entering the input flow path. In somecases, the droplets comprise reaction
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`products from a polymerase chain reaction (PCR).
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`[0038] The sample-containing droplets may flow or be transferred to an intersection region
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`(204), where they may be contacted with an oil-immiscible fluid (e.g., aqueous fluid or air). In
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`somecases, the droplets and oil-immiscible fluid are introduced to the intersection region
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`simultaneously; in some cases, the droplets and the oil-immiscible fluid are introducedto the
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`intersection region sequentially. After the droplets come in contact with the oil-immiscible fluid,
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`they may form a double emulsion, wherein the droplets comprise an aqueous core enveloped or
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`encapsulated by a non-aqueousfluid that is, in turn, surrounded by the oil-immiscible fluid,
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`whichis generally in a continuous phase. In somecases, the oil-immiscible fluid may increase
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`the distance between the droplets (208).
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`[0039] The flow rate of the droplets and the oil-immiscible fluid can be separately controlled.
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`In somecases, the flow of the droplets is controlled by pressure (e.g., vacuum pressure, pump
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`pressure,etc.).
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`[0040] The greater separation may be dueto an increase in fluid speed as fluid approaches and
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`travels inside the output flow path. Further downstream ofthe outlet flow path is at least one
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`detection region. After droplets flow to the detection region (210), the step of detecting a signal
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`(212), such as a fluorescence signal or other signal such as a signal emitted by a radio-isotope,
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`may be carried out. The droplets may be subjected to a stimulus in orderto activate the signal,
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`suchas fluorescentlight or other radiations. For example, the stimulus may be chosen to
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`stimulate emission of fluorescence from one or more fluorescent probes within the droplets. In
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`batch detection applications, the detector and/or the intersection region may be configured to
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`move in a mannerthat allows an optical scan of the detection region by a detector having a
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`smaller field of view than the entire intersection region.
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`[0041] Detected fluorescence may be analyzed to determine whetheror not a particular target
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`nucleotide sequenceis present in the droplets 214. Additional information, including but not
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`limited to an estimate of the numberorfraction of droplets containing a target molecule, the
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`average concentration of target molecules in the droplets, an error margin, and/ora statistical
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`confidence level, also may be extracted from the collected data.
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`[0042] Device
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`[0043] FIG. 3 is a schematic view of an exemplary droplet spacing and/or focusing device that
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`may, optionally, be used in conjunction with a droplet detector/reader. The device may include
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`an input flow path 300, an intersection region 306, an output flow path 314, a radiation source
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`318, a detector 320, and a delivery flow path 324. Emulsified droplets 302 in a non-aqueous
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`continuous fluid 303 may enter the detection system through the input flow path 300. The
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`emulsified droplets may be aqueous droplets dispersed within a non-aqueous(e.g., oil)
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`continuous phase 303. In some cases, an aqueous droplet containing a sample (represented by *)
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`is encapsulated by a layer of non-aqueousfluid. In somecases, the droplets within the input
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`flow path are multiple emulsions. For example, the droplets may be present in a double
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`emulsion and may have an aqueous core enveloped or encapsulated by a non-aqueouslayer and
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`flow in a continuous non-aqueousfluid. In other cases, the droplets are in a triple emulsion, and
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`may have an aqueouscore enveloped or encapsulated by a non-aqueouslayerthat is further
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`enveloped or encapsulated by an aqueouslayer, and the droplets may flow in an aqueous
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`continuous phase. Similarly, the droplets may be a quadruple, quintuple, sextuple, septuple,
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`octuple, or higher-order emulsion. The sample or reaction products may be present in the core of
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`the droplet; however, in some cases the sample or reaction products are present within a
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`particular layer of the emulsion.
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`[0044] Conversely, the droplets may be oil-in-water emulsions. For example, the droplets may
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`have a non-aqueous core and flow in an aqueous continuous phase 303. In this case, an oilis
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`used a focusing /dilution fluid 308. The oil may also form a virtual capillary. The oil-in-water
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`emulsions mayalso be multiple emulsions. In some cases, the droplets may be a double
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`emulsion and have a non-aqueouscore that is enveloped or encapsulated by an aqueouslayer (or
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`oil-immiscible) layer, and the droplets flow in a non-aqueous continuousphase.
`
`[0045] In FIG.3 and throughoutthe present disclosure, the droplets may have different sizes
`
`with respect to the size of the output flow path. In somecases,the ratio of the droplet diameter
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`to the output flow path diameteris less than about 3/1, less than about 2.8/1, less than about
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`2.5/1, less than about 2.2/2, less than about 2.0/1, less than about 1.8/1, less than about 1.5/1, less
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`than about 1.2/1, less than about 1/ 1, less than about 0.8/1, less than about 0.5/1, less than about
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`0.3/1, less than about 0.2/1, or less than about 0.1/1. In somecases,theratio is at least about 3/1,
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`at least about 2.8/1, at least about 2.5/1, at least about 2.2/2, at least about 2.0/1, at least about
`
`1.8/1, at least about 1.5/1, at least about 1.2/1, at least about 1/ 1, at least about 0.8/1, at least
`
`about 0.5/1, at least about 0.3/1, at least about 0.2/1, at least about 0.1/1. In somecases, the ratio
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`is in a range between about 0.1/1 to about 3/ or about 0.5/1 to about 2/1. In a further
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`embodiment, the ratio of the droplet diameter to the output flow path diameteris less than about
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`1/ 1, less than about 0.8/1, less than about 0.5/1, less than about 0.3/1, or less than about 0.2/1.
`
`[0046] Downstream ofthe flow path is at least one intersection region 306. The intersection
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`region 306 may be an intersection of one or more input flow paths 300 and one or more delivery
`
`flow paths 324. In somecases, there are two, three, four, five, six, or even moreintersection
`
`regions. The intersection region may be cross-shaped, as indicated in FIG. 3. In other cases, the
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`intersection is T-shaped, Y-shaped, or other configurations.
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`[0047] As shown in FIG.3, the two paths are substantially perpendicular. However, a variety
`
`of angles can be constructed. The angel may beat least 1 degree, at least 2 degree, at least 5
`
`degree, at least 10 degree, at least 15 degree, at least 20 degree, at least 25 degree, at least 30
`
`degree, at least 35 degree, at least 40 degree, at least 45 degree, at least 50 degree, at least 55
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`degree, at least 60 degree, at least 65 degree, at least 70 degree, at least 75 degree, at least 80
`
`degree, at least 85 degree, at least 90 degree, at least 95 degree, at least 100 degree, at least 105
`
`degree, at least 110 degree, at least 115 degree, at least 120 degree, at least 125 degree, at least
`
`130 degree, at least 135 degree, at least 140 degree, at least 145 degree, at least 150 degree, at
`
`least 155 degree, at least 160 degree, at least 165 degree, at least 170 degree, or at least 175
`
`degree. In some cases, the angel may be about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
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`65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,
`
`170, or 175 degree. In addition, there may be one, two, three, four, five, six, or even more
`
`delivery flow paths, each of which may independently have an angle with respect to the input
`
`flow path. Each delivery flow path may independently contain an oil or an oil-immiscible fluid.
`
`In somecases, an oil or an oil-immiscible fluid is delivered alternatively along a droplet flow
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`path. In somecase, an oil and an oil-immiscible fluid are delivered simultaneously to an
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`intersection region along a droplet flow path through two separate delivery flow paths. In some
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`cases, an oil is delivered consecutively along a droplet flow path through multiple delivery flow
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`paths followed by delivering an oil-immiscible fluid through at least one separate delivery flow
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`path. In somecases, an oil-immiscible fluid is delivered consecutively to a droplet flow path
`
`through multiple delivery flow paths followed by delivering an oil through at least one separate
`
`delivery flow path.
`
`[0048] Uponreachingthe intersection region 306, the droplets may encounter an oil-immiscible
`
`fluid 308 (e.g., an aqueousfluid, air). When the oil-immiscible fluid 308 is an aqueousfluid, the
`
`aqueous fluid may envelop or encapsulate the emulsified droplets 302 to form droplets 310
`
`within a double or other multiple emulsion. The droplets may comprise an aqueouscore,thatis
`
`enveloped or encapsulated by a non-aqueouslayer; and the droplets may travel through a non-
`
`aqueouscontinuous phase 312. The continuous phases 303 and 312 may have the same or
`
`substantially similar composition. The encapsulation may increasethe stability of the droplets
`
`comparedto the droplets in the input flow path 300. The stability of droplets after entering the
`
`output flowpath mayincrease byat least 20%, at least 30%, at least 40%, at least 50%,at least
`
`60%, at least 70%, at least 80%, or at least 90%, compared with the stability of the droplets in
`
`the input path. In addition, the envelopment or encapsulation may prevent release of components
`
`from the aqueousphase of the droplets, which may help preserve the integrity of information
`
`from a prior step (such as a prior PCR amplification).
`
`[0049] The flow rate of droplets 302 and the oil-immiscible fluid 308 may be independently
`
`controlled.
`
`In somecases, the ratio of droplets 302 flow rate/oil-immiscible fluid 308 flow rate
`
`is at least 1/10, 1/9, 1/8, 1/7, 1/6, 1/5, 4, 1/3, %, 1/1, 2/1, 3/1,