Application Data Sheet
`
`Application Information
`
`Application Type::
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`Regwar
`
`Subject Matter::
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`Suggested Group Art Unit::
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`CD—ROM or CD-R?::
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`Sequence submission?::
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`Computer Readable Form (CRF)?::
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`Utility
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`N/A
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`None
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`None
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`No
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`Tit|e::
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`MANIPULATING DROPLET SIZE
`
`Attorney Docket Number::
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`RDT-559/USO1 29168/272
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`No
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`No
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`1 9 Y
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`es
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`No
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`No
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`Request for Early Publication?::
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`Request for Non—Publication?::
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`Suggested Drawing Figure::
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`Total Drawing Sheets::
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`Small Entity?::
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`Petition included?::
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`Secrecy Order in Parent Appl.?::
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`Applicant Information
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`Applicant Authority Type::
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`Status::
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`Given Name::
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`Family Name::
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`Inventor
`
`Full Capacity
`
`Darren
`
`Link
`
`City of Residence::
`
`Lexington
`
`State or Province of Residence::
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`MA
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`Page # 1
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`Initial 07/20/12
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`GEN 1008
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`1
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`Country of Residence::
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`US
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`Street of mailing address::
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`8 Buckman Drive
`
`City of mailing address::
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`Lexington
`
`State or Province of mailing address::
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`MA
`
`Postal or Zip Code of mailing address::
`
`02421
`
`Correspondence Information
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`Correspondence Customer Number::
`
`21710
`
`Representative Information
`
`Representative Customer Number::
`
`21710
`
`Domestic Priority Information
`
`Application::
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`Continuity Type::
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`Parent App|ication::
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`Parent Filing Date:: This Application
`
`61/509837
`
`07/20/11
`
`An application
`claiming the benefit
`under 35 USC
`
`119(e)
`
`Foreign Priority Information
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`Assignee Information
`
`Assignee name::
`
`Raindance Technolgies, Inc.
`
`Street of mailing address::
`
`44 Hartwell Avenue
`
`City of mailing address::
`
`Lexington
`
`State or Province of mailing address::
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`MA
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`Postal or Zip Code of mailing address::
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`02421
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`60617932 V1-WOIkSi[€US-029168/0272
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`Page # 2
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`Initial 07/20/12
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`Attorney Docket No.: RDT—559/US0l 29168/272
`PATENT APPLICATION
`
`MANIPULATING DROPLET SIZE
`
`Related Application
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`The present application claims benefit of and priority to U.S. provisional application
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`serial number 61/509,837, filed July 20, 201 l, the content of which is incorporated by
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`reference herein in its entirety.
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`Field of the Invention
`
`The invention generally relates to methods and systems for manipulating fluidic
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`droplet size.
`
`Background
`
`The ability to precisely manipulate fluidic streams enhances the use and effectiveness
`
`of microfluidic devices. Typically, networks of small channels provide a flexible platform for
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`manipulation of small amounts of fluids. Certain microfluidic devices utilize aqueous
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`droplets in an immiscible carrier fluid. The droplets provide a well—defined, encapsulated
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`microenvironment that eliminates cross contamination and changes in concentration due to
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`diffusion or surface interactions.
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`Microfluidic devices for performing biological, chemical, and diagnostic assays
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`generally include at least one substrate containing one or more etched or molded channels.
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`The channels are generally arranged to form individual fluid circuits, each circuit including a
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`sample fluid channel, an immiscible carrier fluid channel, and an outlet channel. The
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`channels of each circuit may be configured such that they meet at a junction so that droplets
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`of aqueous fluid surrounded by carrier fluid are formed at the junction and flow into the
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`outlet channel. In some cases, the outlet channel of each circuit is connected to a main
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`channel that receives all of the droplets from the different fluidic circuits and flows them to
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`an analysis module. In other cases, the outlet channels connect to exit ports to carry the
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`droplets to a collection vessel.
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`Since each fluidic circuit may have different samples, and because different
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`compositions (e. g., concentration and/or length of nucleic acid) from different samples affect
`
`how droplets form, droplets of different sizes may be produced by each circuit. A problem
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`PATENT APPLICATION
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`with droplets of different sizes flowing through the same channel is that the droplets travel at
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`different velocities. Droplets traveling at different velocities may cause unwanted collisions
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`or unwanted coalescence of droplets in the channel. Thus, it is important that individual
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`fluidic circuits produce droplets of uniform size so that the droplets travel at the same
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`velocity in the channel and do not collide or coalesce in an unwanted manner.
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`Droplets are typically generated one at a time at a junction between an aqueous fluid
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`and an immiscible carrier fluid. Droplet volume and frequency (the number of droplet
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`generated per unit time) are determined by geometrical factors such as the cross—sectional
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`area of the channels at the junction and the fluidic properties such as the fluid viscosities and
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`surface tensions as well as the infusion rates of the aqueous and carrier fluids. To control the
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`volume of the aqueous droplet, within a range, droplet volume can be adjusted by tuning the
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`oil infusion rate through the junction. This is readily achieved with a pressure regulator on
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`the carrier fluid stream. In some cases it is desirable to have multiple junctions operating as
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`separate circuits to generate droplets and have independent control over the oil infusion rates
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`through each circuit. This is readily achieved by using separate pressure regulators for each
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`aqueous stream and each carrier fluid stream. A simpler and lower cost system would have a
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`single carrier oil source at a single pressure providing a flow of carrier oil through each
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`system. The problem with such a system is that in adjusting the pressure to regulate the flow
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`of carrier oil in one circuit the carrier oil in all circuits would be effected and independent
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`control over droplet volume would be compromised. Thus, it is important to have a means
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`whereby at a fixed carrier oil pressure the flow of carrier oil in each of the circuits can be
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`independently controlled to regulate droplet volume.
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`Summary
`
`The invention generally relates to methods and systems for manipulating droplet size.
`
`The invention recognizes that in a fluidic circuit, changing the pressure exerted on the
`
`aqueous phase changes the flow rate of the immiscible carrier fluid. Changing the flow rate
`
`of the immiscible fluid manipulates the size of the droplet. Thus, adjusting pressure, which
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`changes flow rate, adjusts droplet size. Pressure adjustments may be made independent of
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`one another such that the pressure exerted on the aqueous phase in individual fluidic circuits
`
`can be adjusted to produce droplets of uniform size from the different fluidic circuits. In this
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`PATENT APPLICATION
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`manner, droplets produced from different fluidic circuits travel at the same velocity in a main
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`channel and do not collide or coalesce in an unwanted manner.
`
`In certain aspects, the invention provides methods for manipulating droplet size that
`
`involve forming droplets of aqueous fluid surrounded by an immiscible carrier fluid, and
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`manipulating droplet size during the forming step by adjusting pressure exerted on the
`
`aqueous fluid or the carrier fluid. Methods of the invention involve forming a sample droplet.
`
`Any technique known in the art for forming sample droplets may be used with methods of
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`the invention. An exemplary method involves flowing a stream of sample fluid so that the
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`sample stream intersects two opposing streams of flowing carrier fluid. The carrier fluid is
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`immiscible with the sample fluid. Intersection of the sample fluid with the two opposing
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`streams of flowing carrier fluid results in partitioning of the sample fluid into individual
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`sample droplets. The carrier fluid may be any fluid that is immiscible with the sample fluid.
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`An exemplary carrier fluid is oil. In certain embodiments, the carrier fluid includes a
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`surfactant, such as a fluorosurfactant.
`
`Methods of the invention may be conducted in microfluidic channels. As such, in
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`certain embodiments, methods of the invention may further involve flowing the droplet
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`channels and under microfluidic control. Methods of the invention further involve measuring
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`the size of a generated droplet. Any method known in the art may be used to measure droplet
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`size. Preferable methods involve realtime image analysis of the droplets, which allows for a
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`feedback loop to be created so that droplet size may be adjusted in real—time. In certain
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`embodiments, measuring the droplet size is accomplished by taking an image of the droplet
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`and measuring a midpoint of an outline of the droplet image, as opposed to measuring an
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`inside or an outside of the droplet.
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`Another aspect of the invention provides methods for forming droplets of a target
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`volume that include flowing an aqueous fluid through a first channel, flowing an immiscible
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`carrier fluid through a second channel, forming an aqueous droplet surrounded by the carrier
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`fluid, and adjusting resistance in the first or second channels during the forming step to adjust
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`volume of the droplets, thereby forming droplets of a target volume.
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`Another aspect of the invention provides methods for forming substantially uniform
`
`droplets that involve flowing a plurality of different aqueous fluids through a plurality of
`
`different channels, flowing an immiscible carrier fluid through a carrier fluid channel,
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`Attorney Docket No.: RDT—559/US0l 29168/272
`PATENT APPLICATION
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`forming substantially uniform droplets of the different aqueous fluids, each droplet being
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`surrounded by the carrier fluid, by independently adjusting resistance in the different
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`channels.
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`Another aspect of the invention provides microfluidic chips that include a substrate,
`
`and a plurality of channels, in which at least two of the channels include pressure regulators,
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`the pressure regulators being independently controllable. Generally, the plurality of channels
`
`include at least one aqueous fluid channel, at least one immiscible carrier fluid channel, at
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`least one outlet channel, and a main channel. In certain embodiments, the channels are
`
`configured to form microfluidic circuits, each circuit including an aqueous fluid channel, a
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`carrier fluid channel, and an outlet channel. The channels of each circuit meet at a junction
`
`such that droplets of aqueous fluid surrounded by carrier fluid are formed at the junction and
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`flow into the outlet channel. Each outlet channel of each circuit is connected to the main
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`channel. The channels may be etched or molded into the substrate. The channels may be
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`open channels or enclosed channels. Droplets may be collected in a vessel on the device or
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`off of the device.
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`Another aspect of the invention provides droplet systems that include a microfluidic
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`chip that include a substrate, and a plurality of channels, in which at least two of the channels
`
`include pressure regulators, the pressure regulators being independently controllable; and a
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`pressure source coupled to the chip.
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`Other aspects and advantages of the invention are provided in the following
`
`description and claims.
`
`Brief Description of the Drawings
`
`Figure l is a drawing showing a device for droplet formation.
`
`Figure 2 is a drawing showing a device for droplet formation.
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`Figure 3 is a graph showing droplet size sensitivity to changes in aqueous flow rate
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`when using positive displacement pumping.
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`Figure 4 is a graph showing droplet size sensitivity to changes in aqueous flow rate
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`when using pressure driven pumping.
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`Figure 5 shows a diagram of a single fluidic circuit.
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`PATENT APPLICATION
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`Figure 6 is a drawing illustrating that the same volume drop is subject to extreme
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`changes in the lighting but the midpoint is always the same. From left to right, the intensity
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`of the lighting decreases but the midpoint of the outline is always the same.
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`Figures 7A—C provides three graphs that demonstrate the differences in the droplet
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`measuring techniques, and the projected area required to produce 5 pL drops when using the
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`inside, outside and midpoint of a droplet image.
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`Figure 8 is a schematic illustrating measurement of droplet size using the midpoint
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`technique described herein.
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`Figure 9 is a schematic diagram showing a microfluidic interconnect as described in
`
`the Specification, containing a plurality of aqueous fluid ports and an immiscible fluid port
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`for use in methods of the invention.
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`Figure 10 is a schematic diagram showing an apparatus as described in the
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`Specification showing the microfluidic interconnect shown in Figure 9 with a manifold
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`overlay and immiscible fluid storage.
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`Figure ll is a schematic diagram showing the relationship between the microfluidic
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`interconnect of Figure 9 with a microfluidic chip for use in methods of the invention.
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`Detailed Description
`
`The invention generally relates to methods and systems for manipulating droplet size.
`
`In certain aspects, the invention provides methods for manipulating droplet size that involve
`
`forming droplets of aqueous fluid surrounded by an immiscible carrier fluid, and
`
`manipulating droplet size during the forming step by adjusting pressure exerted on the
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`aqueous fluid or the carrier fluid.
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`Droplet Formation
`
`Methods of the invention involve forming sample droplets. In certain embodiments,
`
`the
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`droplets include nucleic acid from different samples. In particular embodiments, each droplet
`
`includes a single nucleic acid template, a single protein molecule or single cell. The droplets
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`are aqueous droplets that are surrounded by an immiscible carrier fluid. Methods of forming
`
`such droplets are shown for example in Link et al. (U.S. patent application numbers
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`Attorney Docket No.: RDT—559/US01 29168/272
`PATENT APPLICATION
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`2008/0014589, 2008/0003142, and 2010/0137163), Stone et al. (U.S. patent number
`
`7,708,949 and U.S. patent application number 2010/0172803), Anderson et al. (U.S. patent
`
`number 7,041,481 and which reissued as RE41,780) and European publication number
`
`EP2047910 to Raindance Technologies Inc. The content of each of which is incorporated by
`
`reference herein in its entirety.
`
`Figure 1 shows an exemplary embodiment of a device 100 for droplet formation.
`
`Device 100 includes an inlet channel 101, and outlet channel 102, and two carrier fluid
`
`channels 103 and 104. Channels 101, 102, 103, and 104 meet at a junction 105. Inlet channel
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`101 flows sample fluid to the junction 105. Carrier fluid channels 103 and 104 flow a carrier
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`fluid that is immiscible with the sample fluid to the junction 105. Inlet channel 101 narrows
`
`at its distal portion wherein it connects to junction 105 (See Figure 2). Inlet channel 101 is
`
`oriented to be perpendicular to carrier fluid channels 103 and 104. Droplets are formed as
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`sample fluid flows from inlet channel 101 to junction 105, where the sample fluid interacts
`
`with flowing carrier fluid provided to the junction 105 by carrier fluid channels 103 and 104.
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`Outlet channel 102 receives the droplets of sample fluid surrounded by carrier fluid.
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`The sample fluid is typically an aqueous buffer solution, such as ultrapure water (e.g.,
`
`18 mega—ohm resistivity, obtained, for example by column chromatography), 10 mM Tris
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`HCl and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any
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`liquid or buffer that is physiologically compatible with enzymes can be used. The carrier
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`fluid is one that is immiscible with the sample fluid. The carrier fluid can be a non—polar
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`solvent, decane (e g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or another oil
`
`(for example, mineral oil).
`
`In certain embodiments, the carrier fluid contains one or more additives, such as
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`agents which reduce surface tensions (surfactants). Surfactants can include Tween, Span,
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`fluorosurfactants, and other agents that are soluble in oil relative to water. In some
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`applications, performance is improved by adding a second surfactant to the sample fluid.
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`Surfactants can aid in controlling or optimizing droplet size, flow and uniformity, for
`
`example by reducing the shear force needed to extrude or inject droplets into an intersecting
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`channel. This can affect droplet volume and periodicity, or the rate or frequency at which
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`droplets break off into an intersecting channel. Furthermore, the surfactant can serve to
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`stabilize aqueous emulsions in fluorinated oils from coalescing.
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`Attorney Docket No.: RDT—559/US0l 29168/272
`PATENT APPLICATION
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`In certain embodiments, the droplets may be coated with a surfactant. Preferred
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`surfactants that may be added to the carrier fluid include, but are not limited to, surfactants
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`such as sorbitan—based carboxylic acid esters (e. g., the "Span" surfactants, Fluka Chemika),
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`including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan
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`monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers
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`(e. g., DuPont Krytox 157 FSL, FSM, and/or FSH). Other non—limiting examples of non—ionic
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`surfactants which may be used include polyoxyethylenated alkylphenols (for example, nonyl—
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`, p—dodecyl—, and dinonylphenols), polyoxyethylenated straight chain alcohols,
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`polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain
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`carboxylic acid esters (for example, glyceryl and polyglycerl esters of natural fatty acids,
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`propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters,
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`etc.) and alkanolamines (e. g., diethanolamine—fatty acid condensates and isopropanolamine—
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`fatty acid condensates).
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`In certain embodiments, the carrier fluid may be caused to flow through the outlet
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`channel so that the surfactant in the carrier fluid coats the channel walls. In one embodiment,
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`the fluorosurfactant can be prepared by reacting the perflourinated polyether DuPont Krytox
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`157 FSL, FSM, or FSH with aqueous ammonium hydroxide in a volatile fluorinated solvent.
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`The solvent and residual water and ammonia can be removed with a rotary evaporator. The
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`surfactant can then be dissolved (e. g., 2.5 Wt %) in a fluorinated oil (e. g., Flourinert (3M)),
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`which then serves as the carrier fluid.
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`Manipulating Droplet Size
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`The invention recognizes that in a fluidic circuit, changing the pressure exerted on the
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`aqueous phase changes the flow rate of the immiscible carrier fluid. Changing the flow rate
`
`of the immiscible fluid manipulates the size of the droplet. Thus, adjusting pressure, which
`
`changes flow rate, adjusts droplet size. Pressure adjustments may be made independently of
`
`each other such that the pressure exerted on the aqueous phase in individual fluidic circuits
`
`can be adjusted to produce droplets of uniform size from the different fluidic circuits. In this
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`manner, droplets produced from different fluidic circuits travel at the same velocity in a main
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`channel and do not collide or coalesce in an unwanted manner. When the pressure is the
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`variable parameter used for control, there is coupling between the aqueous and immiscible
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`PATENT APPLICATION
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`carrier fluid (e. g., oil) channels in an individual circuit. Therefore, any change to the aqueous
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`pressure has an impact on the pressure at the nozzle and in turn affects the flow rate of the
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`immiscible carrier fluid (IMF). For instance, increasing PAq, decreases QIMF and vice—versa.
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`Proper design of the resistances in both the aqueous and immiscible carrier fluid channels
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`controls the degree of coupling that can be expected when making a change to one or more of
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`the input pressures. This in turn controls the sensitivity of the change in drop volume as a
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`function of PA.
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`For comparison, the sensitivity of drop size to a change in flow rate is compared
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`using both a positive displacement pump and a pressure driven system. Figure 3 is a graph
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`showing droplet size sensitivity to changes in aqueous flow rate when using positive
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`displacement pumping. Figure 4 is a graph showing droplet size sensitivity to changes in
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`aqueous flow rate when using pressure driven pumping. Oil was used as the immiscible fluid
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`for these comparisons. Using a similar chip with a similar circuit, a positive displacement
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`pump yields a 10% change in drop volume when changing the flow rate by a factor of two.
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`The pressure driven system yields a 2% change in drop volume for every psi of change in PA.
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`If the pressure was doubled, a 60% change in drop size could be expected when using the
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`pressure driven system. Using a similar circuit, pressure gives 6X better control over the
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`droplet volume when the aqueous channel is adjusted.
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`In certain embodiments, multiple fluidic circuits are used to produce droplets that all
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`flow into a main channel. Proper design of the fluidic circuits, specifically by adjusting the
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`fluidic resistance in both the aqueous and oil channels, controls the degree of influence that
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`adjustments to the aqueous pressure has on each of the circuits, resulting in all of the circuits
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`producing droplets of the same size. Changes in droplet size as a result of changes in pressure
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`and flow rate can be modeled using the below calculations.
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`Figure 5 shows a diagram of a single fluidic circuit for calculation purposes. One of
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`skill in the art will recognize that the calculations shown herein may be applied to multiple
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`fluidic circuits. (A) represents an immiscible carrier fluid channel, (B) represents an aqueous
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`channel, (C) represents a junction of channels (A) and (B) where aqueous phase and
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`immiscible carrier fluid phase meet to form droplets of the aqueous phase surrounded by the
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`immiscible carrier fluid, and (D) represents outlet channel that receives the droplets. PA
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`represents the pressure of the immiscible carrier fluid in the immiscible carrier fluid channel,
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`PB represents the pressure of the aqueous fluid in the aqueous fluid channel, Pc represents the
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`pressure at the junction of channels (A) and (B). PA, PB, and Pc are all greater than 0, and PD
`
`is equal to 0 because channel (D) is open to the atmosphere. QAC represents the flow rate of
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`the immiscible fluid, QBC represents the flow rate of the aqueous fluid, and QCD represents
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`the flow rate of droplets in channel (D). RAC represents the fluidic resistance in the
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`immiscible carrier fluid channel, RBC represents the fluidic resistance in the aqueous channel,
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`and RCD represents the fluidic resistance in the (D) channel. Equations and expressions for
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`QAC and QBC are as follows:
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`PA — PC = QAC(RAC)
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`PB — PC = QBC(RBC)
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`PC = QCD(RCD) = (QAC + QBC)RCD
`
`Equation 1;
`
`Equation 2; and
`
`Equation 3.
`
`Assuming that PA, PB, RAC, BBC, and RCD are known, then the three unknowns are PC, QAC, and
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`QBC. QAC and QBC can be solved for as follows:
`
`QAC =
`
`QBC =
`
`PA(RBC) + (PA— PB)RCD
`
`RAC(RBC) + RCD(RAC + BBC)
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`PB(RAC) + (PA— PB)RCD
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`RAC(RBC) + RCD(RAC + BBC)
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`Equation 3 and
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`Equation 4.
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`The sensitivities of the follow rates (Q) to changes in pressure (P) are determined by
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`obtaining partial derivatives of QAC and QBC with respect to PA and PB, which yields:
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`6QA
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`T =
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`(BBC + RCD)
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`6PA
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`RAC(RBC) + RCD(RAC + BBC)
`
`Equation 5;
`
`6QA
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`RCD
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`T = —
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`Equation 6;
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`6PB
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`RAC(RBC) + RCD(RAC + BBC)
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`PATENT APPLICATION
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`6QB
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`6PB
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`6QB
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`6PA
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`(RAC + RCD)
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`RAC(RBC) + RCD(RAC + RBC)
`
`Equation 7; and
`
`RCD
`
`6QA
`
`RAC(RBC) + RCD(RAC + RBC)
`
`6PB
`
`=
`
`Equation 8.
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`Assuming that P'A = PA + 6PA then:
`
`Q'AC
`
`QAC
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`Q'BC
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`QBC
`
`(RBC + RCD)6PA
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`1+
`
`PA(RBC) + (PA— PB)RCD
`
`Equation 9; and
`
`(RCD)6PA
`
`PB(RAC) - (PA-
`
`PB)RCD
`
`Equation 10.
`
`Similarly, assuming that P"B = PB + 6PB then:
`
`Q"AC
`
`QAC
`
`Q"BC
`
`QBC
`
`(RCD)6PB
`
`PA(RBC) + (PA— PB)RCD
`
`(RAC + RCD)6PB
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`1+
`
`PB(RAC) - (PA- PB)RCD
`
`Equation 11; and
`
`Equation 12.
`
`Substituting chip dPCR 1.3 specifics into the above and assuming PA = PB, thus neglecting PA — PB
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`containing terms yields:
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`Equation 13;
`
`Equation 14;
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`Equation 15; and
`
`Equation 16.
`
`Q'A
`
`QA
`
`=
`
`1 +1.4
`
`6PA
`
`PA
`
`Q'B
`
`6PA
`
`T = -0.45 T
`
`QB
`
`PA
`
`Q"A
`
`QA
`
`Q"B
`
`QB
`
`=
`
`1 -0.36
`
`6PB
`
`PB
`
`=
`
`1 + 3
`
`6PB
`
`PB
`
`The results in Figure 4 show that changing PA from 28 psi to 30 psi results in QBC
`
`going from 577 uL/hr to 558 uL/hr, — 3.3% change. The above model predicts a — 3.1%
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`change in QB1which is in agreement with the actually results data.
`
`In certain embodiments, the system may be configured such that the circuits produce
`
`droplets of different size to allow for controlled droplet coalescence in the main channel. The
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`fluidic circuits are arranged and controlled to produce an interdigitation of droplets of
`
`different sizes flowing through a channel. Such an arrangement is described for example in
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`Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and
`
`2010/0137163) and European publication number EP20479 10 to Raindance Technologies
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`Inc. Due to size Variance, the smaller droplet will travel at a greater Velocity than the larger
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`droplet and will ultimately collide with and coalesce with the larger droplet to form a mixed
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`droplet.
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`Another benefit of the added resistance in both channels next to the nozzle occurs
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`during priming. Simultaneous arrival of both the aqueous and carrier liquids is difficult to
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`produce reliably. If the carrier fluid enters the aqueous channel and travels all the way back
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`into the filter elements, the aqueous and carrier liquids begin to mix and emulsify before the
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`nozzle. This mixing interference causes significant variability in the size of the generated
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`droplets. The added resistance next to the nozzle eliminates the mixing interference by
`
`creating a path of relatively high resistance without emulsifying features that are in the filter.
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`Therefore, if the carrier fluid arrives at the nozzle first it will travel both into the aqueous
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`resistor and towards the outlet of the chip. The outlet of the chip has a resistance that is much
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`smaller than the aqueous resistor and therefore the majority of the carrier fluid will flow in
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`that direction. This gives the aqueous liquid time to reach the nozzle before the carrier fluid
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`enters the filter feature.
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`Droplet Measurement
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`The volume of an individual droplet is measured using real—time image analysis. This
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`in turn is fed back into a control loop where a known projected area is targeted and equal to a
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`given droplet volume. Microfluidic chips are calibrated using a 3 point reference emulsion of
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`know volumes to generate calibration curves for each channel. The idea is that the midpoint
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`of the outline of a projected droplet image is always the same regardless of the lighting. This
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`demonstrated in figure 6, which is a drawing illustrating that the same volume drop is subject
`
`to extreme changes in the lighting but the midpoint is always the same. From left to right, the
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`intensity of the lighting decreases but the midpoint of the outline is always the same.
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`In contrast to determining the projected area of the inside of the drop, which is difficult
`
`due to chip and lighting imperfections and variability, or the outside of the drop, which is
`
`also quite sensitive to lighting and chip imperfections, methods of the invention use the
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`midpoint of the outline of a projected droplet image, which is always the same regardless of
`
`the lighting and chip imperfections. Using the midpoint “flattens out” the imperfections and
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`is significantly less sensitive to outside influences on projected drop size. Figures 7A—C
`
`provides three graphs that demonstrate the differences in the droplet measuring techniques,
`
`and the projected area required to produce 5 pL drops when using the inside, outside and
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`midpoint of a droplet image. Finding both the outside and inside projected area allows you
`
`calculate the outside and inside diameters. Calculating the average of the outside and inside
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`diameters gives you the midpoint diameter. From there an estimated projected area is
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`calculated from the midpoint diameter (See Figure 8).
`
`Nucleic Acid Target Molecules
`
`One of skill in the art will recognize that methods and systems of the invention are
`
`not limited to any particular type of sample, and methods and systems of the invention may
`
`be used with any type of organic, inorganic, or biological molecule. In particular
`
`embodiments the droplets include nucleic acids. Nucleic acid molecules include
`
`deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). Nucleic acid molecules can be
`
`synthetic or derived from naturally occurring sources. In one embodiment, nucleic acid
`
`molecules are isolated from a biological sample containing a variety of other components,
`
`such as proteins, lipids and nontemplate nucleic acids. Nucleic acid template molecules can
`
`be obtained from any cellular material, obtained from an animal, plant, bacterium, fungus, or
`
`any other cellular organism. In certain embodiments, the nucleic acid molecules are obtained
`
`from a single cell. Biological samples for use in the present invention include viral particles
`
`or preparations. Nucleic acid molecules can be obtained directly from an organism or from a
`
`biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid,
`
`seminal fluid, saliva, sputum, stool and tissue. Any tissue or body fluid specimen may be
`
`used as a source for nucleic acid for use in the invention. Nucleic acid molecules can also be
`
`isolated from cultured cells, such as a primary cell culture or a cell line. The cells or tissues
`
`from which template nucleic acids are obtained can be infected with a virus or other
`
`intracellular pathogen. A sample can also be total RNA extracted from a biological specimen,
`
`a cDNA library, viral, or genomic DNA.
`
`Generally, nucleic acid can be extracted from a biological sample by a variety of
`
`techniques such as those described by Maniatis, et al., Molecular Cloning: A Laboratory
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`Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Nucleic acid molecules may be
`
`single—stranded, double—stranded, or double—stranded with single—stranded regions (for
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`example, stem— and loopstructures).
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`Methods of the invention further involve amplifying a target nucleic acid(s) in a
`
`droplet. Amplification refers to production of additional copies of a nucleic acid sequence
`
`and is generally carried out using polymerase chain reaction or other technologies well
`
`known in the art
`
`(e. g., Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor
`
`Press, Plainview, N.Y. [1995]). The amplification reaction may be any amplification reaction
`
`known in the art that amplifies nucleic acid molecules, such as polymerase chain reaction,
`
`nested polymerase chain reaction, polymerase chain reaction—single strand conformation
`
`polymorphism, ligase chain reaction (Barany F. (1991) PNAS 88: 189-193; Barany F. (1991)
`
`PCR Methods and Applications 1:5—16), ligase detection reaction (Barany F. (1991) PNAS
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`88: 189-193), strand displacement amplification and restriction fragments length
`
`polymorphism, transcription based amplification system, nucleic acid sequence—based
`
`amplification, rolling circle amplification, and hyper—branched rolling circle amplification.
`
`In certain embodiments, the amplification reaction is the polymerase chain reaction.
`
`Polymerase chain reaction (PCR) refers to methods by K. B. Mullis (U.S. patent numbers
`
`4,683,195 and 4,683,202, hereby incorporated by reference) for increasing concentration of a
`
`segment of a target sequence in a mixture of genomic DNA without cloning or purification.
`
`The process for amplifying the target sequence includes introducing an excess of
`
`oligonucleotide primers to a DNA mixture containing a desired target sequence, followed by
`
`a precise sequence of thermal cycling in the presence of a DNA polymerase. The primers are
`
`complementary to their respective strands of the double stranded target sequence.
`
`To effect amplification, primers are annealed to their complementary sequence within
`
`the target molecule. Following annealing, the primers are extended with a polymerase so as
`
`to form a new pair of complementary strands. The steps of denaturation, primer annealing
`
`and polymerase extension can be repeated many times (i.e., denaturation, annealing and
`
`extension constitute one c