Patent Owner RainDance Technologies, Inc.
`
`Oral Argument
`
`10x Genomics, Inc. v. RainDance Technologies, Inc.
`
`Case lPR2015-01158
`
`U.S. Patent No. 8,658,430
`
`September 27, 2016
`
`

`
`Overview Of The ’43O Patent
`
`

`
`The ’430 Patent: Multiple Droplet Forming Junctions
`
`1. A method for droplet formation, the method comprising
`the steps of:
`providing a each in its own
`aqueous fluid charmel in fluid communication with one
`or more immiscible carrier fluid channels;
`forming droplets ofaqueous fluid surrounded by an immis-
`cible carrier fluid in the aqueous fluid channels;
`applying a same constant pressure to the carrier fluid in
`each of the immiscible carrier fluid channels; and
`adjusting pressure in one or more of the aqueous fluid
`channels, thereby to produce droplets ofaqueous fluid in
`one or more outlet fluid charmels.
`
`(cited in Paper 21, Patent Owner's Response, p. 14).
`
`Ex. 1001 (430 Patent), claim 1
`
`

`
`Dr. Huck: ’430 Patent Requires Multiple Junctions
`
`Q. All right. And so you understand that the claims of the
`'43O patent require multiple droplet-forming junctions,
`right?
`
`A.
`
`I understand that.
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 154:1—4
`
`(cited in Paper 21, Patent Owner's Response, p. 15).
`
`

`
`Dr. Huck’s View Of The "Method of the ’430 Patent”
`
`Figure 1: Method of the ’430 Patent
`
`Aqueous fluid flow
`
`Immiscible
`carrier fluid flow
`
`lmmiscible
`carrier fluid flow
`
`Immiscible
`carrier fluid flow
`
`Aqueous fluid flow
`
`lmmiscible
`carrier fluid flow
`
`Key for Fig. 1
`
`- Aqueous fluid 2
`
`I Carrier fluid
`
`- Aqueous fluid 1
`
`Ex. 1002 (Huck Dec/.), Fig. 1.
`
`

`
`A Microfluidic Circuit
`
`Carrier fluid channel
`
`A
`
`1
`
`T
`
`C
`
`Outlet channel
`
`D
`
`B
`
`Sample channel
`FIG. 5
`
`(cited in Paper 21, Patent Owner's Response, p. 10).
`
`Ex. 1001 (430 Patent), Fig. 5
`
`

`
`Two Microfluidic Circuits
`
`(cited in Paper 21, Patent Owner's Response, p. 10).
`
`EX. 1001 (430 Patent), Fig. 5
`
`

`
`Two Coupled Microfluidic Circuits
`
`
`
`carrier fluid
`
`(cited in Paper 21, Patent Owner's Response, p. 10).
`
`EX. 1001 (430 Patent), Fig. 5
`
`

`
`’430 Patent: The Problem With Coupled Circuits
`
`To control the Volume of the aqueous droplet,
`Within a range, droplet Volume can be adjusted by tuning the
`oil infusion rate through the junction. This is readily achieved
`with a pressure regulator on the carrier fluid stream. In some
`cases it is desirable to have multiple junctions operating as
`separate circuits to generate droplets and have independent
`control over the oil infusion rates through each circuit. This is
`readily achieved by using separate pressure regulators for
`each aqueous stream and each carrier fluid stream. A simpler
`and lower cost system would have a single carrier oil source
`at a single pressure providing a flow of carrier oil through
`
`each s stem.
`
`Thus, it is important to have a means whereby at a
`fixed carrier oil pressure the flow of carrier oil in each of the
`circuits can be independently controlled to regulate droplet
`Volume.
`
`(cited in Paper 21, Patent Owner's Response, pp. 8-9).
`
`Ex. 1001 (430 Patent) at 1:59-2:10
`
`

`
`’430 Patent: "Same Constant Pressure” On All Oil Channels
`
`1. A method for droplet formation, the method comprising
`the steps of:
`providing a plurality of aqueous fluids each in its own
`aqueous fluid charmel in fluid communication with one
`or more immiscible carrier fluid channels;
`forming droplets ofaqueous fluid surrounded by an immis-
`cible carrier fluid in the aqueous fluid charmels;
`
`'—$
`
`adjusting pressure in one or more of the aqueous fluid
`channels, thereby to produce droplets ofaqueous fluid in
`one or more outlet fluid channels.
`
`(cited in Paper 21, Patent Owner's Response, p. 14).
`
`Ex. 1001 (430 Patent), claim 1
`
`

`
`The Technique Of The ’430 Patent
`
`the size of the dro let.
`
` Changing the flow rate of the immiscible fluid mani ulates
`
`Pres sure adjustments
`may be made independent of one another such that the pres-
`sure exerted on the aqueous phase in individual fluidic cir-
`cuits can be adjusted to produce droplets ofuniform size from
`the different fluidic circuits.
`
`(cited in Paper 21, Patent Owner's Response, p. 26).
`
`Ex. 1001 (430 Patent) at 5:15-20
`
`

`
`The Technique Of The ’430 Patent
`
`When the pressure
`is the Variable parameter used for control, there is coupling
`between the aqueous and immiscible carrier fluid (e.g., oil)
`channels in an individual circuit. Therefore, any change to the
`aqueous pressure has an impact on the pressure at the nozzle
`and in turn affects the flow rate of the immiscible carrier fluid
`
`(IMF). For instance, increasing PAQ, decreases QIMF and Vice-
`Versa. Proper design of the resistances in both the aqueous
`and immiscible carrier fluid channels controls the degree of
`coupling that can be expected when making a change to one
`or more of the input pressures. This in turn controls the
`sensitivity of the change in drop Volume as a function of PA.
`
`(cited in Paper 21, Patent Owner's Response, p. 11).
`
`Ex. 1001 (430 Patent) at 5:34-45
`
`

`
`Dr. Huck: Aqueous Pressure Adjustments Changes The Oil Flow Rate
`
`Q. Okay. And- you understand that you have
`the aqueous fluid channels coupled to the oil channels,
`right?
`
`A. That is correct.
`
`Q. Okay. And there's a constant pressure on all the oil
`channels, right?
`
`A. That's What you do.
`
`«.9
`
`Okay. And for the reasons We've been discussing today,
`
`Ex. 2015 (Apri/13, 2016 Huck Dep. Tr.) at 177:11—178:1
`
`(cited in Paper 21, Patent Owner's Response, pp. 25-26).
`
`

`
`’430 Patent: Claims 5-9
`
`5. The method of claim 1, wherein the droplets comprising
`a first aqueous fluid and the droplets comprising a second
`aqueous fluid are substantially-
`6. The method of claim 1, wherein the droplets comprising
`a first aqueous fluid are a- than the droplets
`comprising a second aqueous fluid.
`7. The method of claim 5 or 6, further comprising the step
`of— of the droplets in one or more of the
`aqueous fluid channels.
`8. The method of claim 7, further comprising the step of
`— applied to the carrier fluid and/or the
`pressure applied to the one or more of the aqueous fluid
`channels based on the detecting step.
`9. The method of claim 6, further comprising the step of
`— applied to a first of the aqueous fluid
`channels based on the detecting step, thereby to-
`— in the first aqueous fluid channel and not
`in other aqueous fluid channels.
`
`(cited in Paper 21, Patent Owner's Response, pp. 15-16).
`
`Ex. 1001 (430 Patent), claims 5-11
`
`

`
`Dr. Huck: Flow Rate Control Is Not Pressure Control
`
`Q. Okay. But with the‘ pump, you
`control the flow rate‘?
`
`A.
`
`Ibelieve, typica11y,—
`
`Q. And so in a flow rate-controlled device, you're making
`sure that a fixed volume of liquid travels through the
`device in any given time interval; is that correct?
`
`A. That is correct. You control the flow rates through the
`device.
`
`Q. Okay. And can that be contrasted with pressure control?
`
`A 1think
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 126:9-22
`
`(cited in Paper 21, Patent Owner's Response, p. 27).
`
`

`
`Dr. Huck: Flow Rate Control Is Not The ’430 Patent
`
`Q.
`
`I'd like to direct you back to Claim 1 of the ’430 patent.
`Now, have you or anyone in your lab ever performed the
`method of Claim 1?
`
` in the sense that We would adjust, for example,
`
`the flow rate in one or more of the aqueous channels
`rather than the pressure in one or more of the aqueous
`fluid channels, although the end result would be the
`same, that We would have a plurality of aqueous fluids
`that would form droplets and Where We can adjust the
`flow rate in one or more of the aqueous fluid channels to
`produce droplets of aqueous fluid.
`
`Ex. 2017 (August 23, 2016 Huck Dep. Tr.) at 337:14—338:2
`
`(cited in Paper 37, Motion for Observations, pp. 3-4).
`
`

`
`Anticipation
`
`17
`
`

`
`’430 Patent: Claim 1
`
`1. A method for droplet formation, the method comprising
`the steps of:
`providing a plurality of aqueous fluids each in its own
`aqueous fluid channel in fluid communication with one
`or more immiscible carrier fluid channels;
`forming droplets ofaqueous fluid surrounded by an immis-
`cible carrier fluid in the aqueous fluid charmels;
`applying a same constant pressure to the carrier fluid in
`each of the immiscible carrier fluid channels; and
`
`
`
`(cited in Paper 21, Patent Owner's Response, p. 14).
`
`Ex. 1001 (430 Patent), claim 1
`
`

`
`Dr. Huck: Link Does Not Disclose Same Constant Pressure
`
`Q. Would a person of ordinary skill in the art following the
`teachings of Link inherently use the same constant
`pressure for the carrier channel and adjust the pressure
`in aqueous channel to produce droplets?
`
`A. &. Link says controlling the pressure difference
`between the oil and the Water source, and, to me, the
`person Would then know What the options are to control
`that pressure difference.
`
`Ex. 2017 (August 23, 2016 Huck Dep. Tr.) at 375:4—12 (objection omitted)
`
`(cited in Paper 37, Motion for Observations, p. 1).
`
`

`
`Petitioner's Observation Response: Link Does Not Anticipate
`
` This is relevant because ll
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`
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`
`that Link teaches “controlling the pressure difference between the oil and Water
`
`source” and that a POSA would have immediately recognized that the options to
`
`do so include “(1) altering the aqueous fluid pressure while keeping the carrier
`
`fluid pressure constant, (2) altering the carrier fluid pressure while keeping the
`
`aqueous fluid pressure constant, or (3) altering both the aqueous fluid and carrier
`
`fluid pressure.” EXIOOZ, 1144; EX1036, 1133. Dr. Huck’s testimony makes clear that
`
`a POSA would have chosen from the three options taught in Link. That Link
`
`teaches two options for modifying droplet size in addition to the claimed method,
`
`does not negate that Link teaches a POSA the claimed method, and therefore
`
`anticipates the claims.
`
`Paper 40 (Response to Motion for Observations), pp. 1-2.
`
`

`
`Link 1] 166: Does Not Disclose Same Constant Pressure
`
`the inlet module can also be
`[0166] The pressure at
`regulated by adjusting the pressure on the main and sample
`inlet channels, for example, with pressurized syringes feed-
`ing into those inlet channels.
`
`
`
`
`
`Alternatively, a Valve may be placed at or
`coincident to either the inlet module or the sample inlet
`channel connected thereto to control the flow of solution into
`
`
`
`the inlet module, thereby controlling the size and periodicity
`of the droplets. Periodicity and droplet Volume may also
`depend on channel diameter, the Viscosity of the fluids, and
`shear pressure.
`
`(cited in Paper 21, Patent Owner's Response, p. 36).
`
`Ex. 1004 (Link), 7] 166
`
`

`
`The Board's Institution Decision
`
`Our determination is based in part 011 the following disclosure in Link,
`
`which Petitioner relies upon to teach the “constant pressure” step:
`
`The pressure at the inlet module can also be regulated by
`adjusting the pressure on the main and sample inlet channels,
`for example, with pressurized syringes feeding into those inlet
`channels. By controlling the pressure difference between the
`oil and water sources at the inlet module, the size and
`
`periodicity of the droplets generated may be regulated.
`
`zsi; Pet. 22. This passage discloses “adjusting the pressure on
`
`the 1r1ain and sample inlet channels,” which in the parlance of the ’430
`
`patent means adjusting the pressure on the carrier fluid and aqueous fluid
`
`channels. We are not persuaded that this passage teaches "the exact
`
`opposite" of the pressure limitations of claim 1. as argued by Patent Owner.
`
`Preliin Resp. 25. Link teaches that droplet size is regulated by "controlling
`
`the pressure difference between the oil and water sources at the inlet
`
`module." Ia’. (emphasis added).
`
`
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`
`Paper 13 (Institution Decision) at p. 11
`
`(cited in Paper 21, Patent Owner's Response, pp. 19
`
`

`
`Link 1] 166: Does Not Disclose Same Constant Pressure
`
`
`
`oflchannel
`
`hfletchannel
`
`foraqueous
`
`sampm
`
`(cited in Paper 21, Patent Owner's Response, p. 20).
`
`Ex. 1004 (Link), Fig. 1
`
`

`
`Dr. Huck: Link 1] 166 Leaves "Open” Whether It Uses A Single Oil Source
`
`Q.
`
`A.
`
`Is there anywhere else in the Link reference, other than
`Paragraph 166, that you contend discloses using a single syringe
`to feed multiple oil lines that go into a single droplet-forming
`microfluidic junction?
`
`I believe that Link leaves this — leaves this open to the person of
`skill in the art in how to set up the device. He mentions
`pressurized syringes. I think people in the literature would
`typically use one — for this specific geometry, which is a flow-
`focusing nozzle, Would typically use a syringe for oil and a syringe
`for the aqueous line, and the oil is split and then converges back
`into the — into the nozzle. So I, therefore, think it is open for the
`people Working with these devices to — to construct it in the way
`they Want.
`
`=93
`
`And it's left open in the Link reference, right?
`
`A.
`
`I don't remember looking specifically for examples where he said
`how you should tackle this, but in 166 he mentioned the — using
`the pressurized syringe to feed the inlet channels.
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 197:19—198:20
`
`(cited in Paper 21, Patent Owner's Response, p. 39).
`
`

`
`The Board's Institution Decision
`
`Our determination is based in part on the following disclosure in Link,
`
`which Petitioner relies upon to teach the “constant pressure” step:
`
`The pressure at the inlet module can also be regulated by
`adjusting the pressure on the main and sample inlet channels,
`for example, with pressurized syringes feeding into those inlet
`channels. By controlling the pressure difference between the
`oil and water sources at the inlet module, the size and
`
`periodicity of the droplets generated may be regulated.
`
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`
`Paper 13 (Institution Decision) at pp. 10-11
`
`(cited in Paper 21, Patent Owner's Response, p. 40).
`
`

`
`Dr. Huck: The Main Channel Is Not An Oil Channel
`
`{IT
`comgséemce w—aeLEA~_
`MDULE
`MODULE
`
`IN ST
`MO LE
`
`
`
`-
`
`‘ uarmnv
`
` couecnow sm=m"c; WASTE
`
`momma
`MODULE
`MODULE
`
`Fig. 1
`
`Ex. 2011 (Huck markup of Link, Fig 1)
`
`(cited in Paper 21, Patent Owner's Response, p. 40).
`
`Carrier fluid channels
`
`

`
`Dr. Huck: The Main Channel Is Not An Oil Channel
`
`Q. All right. I'm handing you a copy of Figure 1 of the Link
`reference. Okay. Where is the main channel in Figure 1
`
`of the Link reference using the description we just
`looked at in Paragraph 103?
`
` So after you make your droplets, then there is a
`
`main channel, where all kinds of things happen, and
`then at the end there's an outlet, or multiple outlets.
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 200:5—15
`
`(cited in Paper 21, Patent Owner's Response, p. 40).
`
`

`
`Dr. Huck: The Main Channel Is Not An Oil Channel
`
`Q. Okay. NoW1et's take a look at 166. 166 is, again,
`referring to the main Channel, in the first sentence,
`right?
`
`A. Yes.
`
`Q. Okay. And that main channel that's being referred to
`there, again, that is the main channel that you identify
`in Exhibit 2011?
`
`A:
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 205:21—206:6
`
`(cited in Paper 21, Patent Owner's Response, p. 40).
`
`

`
`Dr. Huck: Link Figure 1 Does Not Disclose Same Constant Pressure
`
`Q.
`
`Is it your contention that, in Figure 1 of Link, the
`pressure that's applied to all of the four admissible
`carrier fluid channels is the same?
`
`A-—
`
`Q. But not necessarily?
`
`A-—
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 206:18—207:2
`
`(cited in Paper 21, Patent Owner's Response, p. 39).
`
`

`
`Dr. Huck: Link Figure 1 Does Not Disclose Same Constant Pressure
`
`Q. Okay. So you're not saying in Paragraph 99 of your
`declaration that Link, Figure 1, discloses that all of the
`[immiscible] oil channels that are feeding into the
`system and providing oil for the generation of droplets
`are coming from a single source, right‘?
`
`A-—It can come
`from the same single source, and it can come from
`multiple sources, but it's the same carrier fluid.
`
`Ex. 2015 (April 13, 2016 Huck Dep. Tr.) at 212:11-20
`
`(cited in Paper 21, Patent Owner's Response, p. 39).
`
`

`
`Link: Discloses The Use Of Multiple Oil Sources
`
`

`
`Dr. Huck: Link Only Teaches Constant Flow
`
`Q. Okay. So can — why don't you do this: Why don't you identify
`for me, by paragraph number, where you believe the Link
`reference — and when I say paragraph number, I mean
`paragraph of the Link reference, which paragraph of the Link
`reference expressly discloses the applying a same constant
`pressure limitation, putting aside other literature references
`and what someone of skill in the art might know. I just want to
`know where it's expressly disclosed in the Link reference,
`which paragraph number you're relying upon for that.
`A. Well, I think in 132,othere is a mention of providin a constant
`flow of oil. And I combined that with Paragraph 10, which has
`multiple inlet modules. And that I believe — I think in
`combination with the fact that the channels areén fluid
`communicatior9and teachings in Paragraph 110 and 11,6about
`the continuous fluid stream, I believe they, together, show the
`POSA that there is o_rthere can be a constant flow in all the oil
`channels.
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 222:10—223:10
`
`(cited in Paper 21, Patent Owner's Response, p. 57).
`
`

`
`Dr. Huck: Link TH] 131-132 Discloses Single Syringe As Oil Source
`
`46.
`
`Link discloses that the microfluidic device may provide a “constant
`
`flow” of carrier fluid into the immiscible carrier fluid channel.
`
`(GENl004,
`
`M0132], [0010], [0()38], [O088], [0110]-[0112], [0164]-[0l66], [0173], claim 16.)
`
`fl Milt‘.
`
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`
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`understood the pressure applied to the aqueous fluid channels by the syringe would
`
`be distributed equally, i.e., the same, across the carrier fluid channels to which the
`
`pressure is applied. Further, as discussed in §VII above, a POSA would have
`
`understood that “applying a constant pressure” encompasses at least generating a
`
`constant flow of carrier fluid in a carrier fluid channel by applying pressure from a
`
`single source, such as a syringe, wherein the pressure is distributed equally across
`
`all channels to which the pressure is applied. Therefore Link discloses applying a
`
`same constant pressure to the carrier fluid in the immiscible carrier fluid channel.
`
`(cited in Paper 21, Patent Owner's Response, p. 57).
`
`Ex. 1002 (Huck Dec/.), 1] 46
`
`

`
`
`
`Link 111] 131-32
`
`
`
`[0132]
`
`Since the flow is three dimensional, under this
`
`design surface wetting elfects are minimized.
`
`
`
`The high resolution part of the nozzle can be made out of a
`small bore tubing or a small, simple part molded or stamped
`from an appropriate material (Teflon®, plastic, metal, etc).
`If necessary, the nozzle itself could be formed into the tip of
`the ferrule using post mold processing such as laser ablation
`or drilling.
`
`[0131] The present invention provides compositions and
`methods for forming sample droplet emulsions on a microf-
`luidic substrate. The present invention also provides embed-
`ded microfluidic nozzles.
`
`
`
`
`
`FIG. 2, Panels A
`
`and B, show dual and single oil versions of the nozzle
`concept using a small ferrule for the nozzle section. FIG. 2,
`Panels C and D, show the same Nozzles made directly out
`of small bore tubing (the “nozzle” runs the entire length of
`the tubing). Both designs can form droplets identically,
`although the pressure drop will be higher for the tube based
`nozzle (bottom). FIG. 3 shows the expansion of the nozzle
`ferrule concept shown in FIGS. 2A and 2B. The tube based
`nozzles (FIG. 2C, 2D) function identically to this, except the
`“nozzle” runs the entire length of the tube instead of having
`a short transition. The ability to form droplets is identical in
`both cases. FIG. 4 shows the expansion of the nozzle section
`contained in the ferrule. The tee design in FIG. 2D has been
`built and tested, with a cross-section cut of this design
`shown in FIG. 5. FIG. 5A shows the operation of the nozzle
`in Aspiration Mode and FIG. 5B shows the operation of the
`nozzle in Injection Mode. The droplets formed are approxi-
`mately 45 um in diameter, and were formed from PCR mix
`(210 ul/l1r) and SpectraSyn-10 (600 ul/hr). Other tests have
`been demonstrated with Spectrasyn-2 and PCR mix. The
`droplets are traveling in 300 urn wide><260 urn deep chan-
`nels. The Nozzle tube used was 100 um in diameter, and the
`
`fluids used were PCR Mix and Spectrasyn-10 with surfac-
`tant.
`
`(cited in Paper 21, Patent Owner's Response, pp. 31-33).
`
`Ex. 1004 (Link), 77 131-132
`
`

`
`Dr. Huck: Link 1| 131 Discloses A Droplet Collection Syringe
`
`Q. Okay. So that syringe that's being referred to in
`‘ that's the storage syringe for collecting
`the droplets, right?
`
`AZ-——
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 186:21—187:3
`
`(cited in Paper 21, Patent Owner's Response, p. 32).
`
`

`
`Dr. Huck: Link 1| 132 Discloses A Droplet Collection Syringe
`
`Q. Just confirm for me, the syringes referred to in
`‘ that's for collecting the droplets, right?
`
`A. The syringe that We're talking about here, which is
`different from syringes that are discussed elsewhere, is
`one for-
`
`Ex. 2015 (April13, 2016 Huck Dep. Tr.) at 188:6-12
`
`(cited in Paper 21, Patent Owner's Response, p. 32).
`
`

`
`Dr. Huck: Syringe ls Attached To Droplet Outlet
`
`Ex. 2010 (Huck markup of 430 Patent, Fig. 6A)
`
`(cited in Paper 21, Patent Owner's Response, p. 32).
`
`37
`
`

`
`Link 1] 38 Does Not Disclose Same Constant Pressure
`
`[0038] The continuous phase can be a non-polar solvent.
`The continuous phase can be a fluorocarbon oil. The con-
`tinuous phase can further include one or more additives.
`Preferably, the additive is a fluorosurfactant. More prefer-
`
`, the fluorosurfactant is a erfluorinated ol ether.
`
`(cited in Paper 21, Patent Owner's Response, pp. 28-29).
`
`Ex. 1004 (Link), 77 38
`
`

`
`Link 1] 88 Does Not Disclose Same Constant Pressure
`
`[0088] The microfluidic device of the present invention
`includes one or more analysis units. An “analysis unit” is a
`microsubstrate, e.g., a microchip. The terms microsubstrate,
`substrate, microchip, and chip are used interchangeably
`herein. The analysis unit includes at least one inlet channel,
`at least one main channel, at least one inlet module, at least
`one coalescence module, and at least one detection module.
`The analysis unit can further includes one or more sorting
`modules. The sorting module can be in fluid communication
`with branch channels which are in fluid communication with
`
`one or more outlet modules (collection module or waste
`module). For sorting applications, at least one detection
`module cooperates with at least one sorting module to divert
`flow Via a detector-originated signal. It shall be appreciated
`that the “modules” and “channels” are in fluid communica-
`
`tion with each other and therefore may overlap; i.e., there
`may be no clear boundary where a module or channel begins
`or ends. A plurality of analysis units of the invention may be
`combined in one device. The analysis unit and specific
`modules are described in further detail herein.
`
`(cited in Paper 21, Patent Owner's Response, p. 29).
`
`Ex. 1004 (Link), 77 88
`
`

`
`Link 1] 110 Does Not Disclose Same Constant Pressure
`
`A liquid that does not
`contain sample molecules, cells or particles can be intro-
`duced into a sample inlet well or channel and directed
`through the inlet module, e.g., by capillary action, to hydrate
`and prepare the device for use. Likewise, buficer or oil can
`also be introduced into a main inlet region that communi-
`cates directly with the main channel to purge the device
`(e.g., or “dead” air) and prepare it for use. If desired, the
`pressure can be adjusted or equalized, for example, by
`adding buffer or oil to an outlet module.
`
`(cited in Paper 21, Patent Owner's Response, pp. 29-30).
`
`Ex. 1004 (Link), 7] 110
`
`

`
`Link 1] 111-12 Does Not Disclose Same Constant Pressure
`
`[0111] As used herein, the term “fluid stream” or “fluidic
`stream” refers to the flow of a fluid, typically generally in a
`specific direction. The fluidic stream may be continuous
`and/or discontinuous. A “continuous” fluidic stream is a
`
`fluidic stream that is produced as a single entity, e. g., if a
`continuous fluidic stream is produced from a channel, the
`fluidic stream, after production, appears to be contiguous
`with the channel outlet. The continuous fluidic stream is also
`
`referred to as a continuous phase fluid or carrier fl11id. The
`continuous fluidic stream may be laminar, or turbulent in
`some cases.
`
`Similarly, a “discontinuous” fluidic stream is a
`[0112]
`fluidic stream that is not produced as a single entity. The
`discontinuous fluidic stream is also referred to as the dis-
`
`persed phase fluid or sample fluid. A discontinuous fluidic
`stream may have the appearance of individual droplets,
`optionally surrounded by a second fluid. A “droplet,” as used
`herein, is an isolated portion of a first fluid that completely
`surrounded by a second fluid. In some cases, the droplets
`may be spherical or substantially spherical; however,
`in
`other cases, the droplets may be non-spherical, for example,
`the droplets may have the appearance of “blobs” or other
`irregular shapes, for instance, depending on the external
`environment. As used herein, a first entity is “surrounded”
`by a second entity if a closed loop can be drawn or idealized
`around the first entity through only the second entity. The
`dispersed phase fluid can include a biological/chemical
`material. The biological/chemical material can be tissues,
`cells, particles, proteins, antibodies, amino acids, nucle-
`otides, small molecules, and pharmaceuticals. The biologi-
`
`(cited in Paper 21, Patent Owner's Response, pp. 30-31).
`
`Ex. 1004 (Link), 1777 111-112
`
`

`
`Link 1] 164-65 Do Not Disclose Same Constant Pressure
`
`
`
`However, other methods may also be
`used, alone or in combination with pumps and valves, such
`as electro-osmotic flow control, electrophoresis and dielec-
`Uophorefls (Fuhvyen Ekjence 156, 910 (1974fi
`IA and
`Harrison, Analytical Chemistry 69, 1564 (1997); Fiedler, et
`al. Anabitical C/aemistry 70, 1909-1915 (1998); US. Pat.
`No. 5,656,155). Application of these techniques according to
`the invention provides more rapid and accurate devices and
`methods for analysis or sorting, for example, because the
`sorting occurs at or in a sorting module that can be placed
`at or immediately after a detection module. This provides a
`shorter distance for molecules or cells to travel, they can
`move more rapidly and with less turbulence, and can more
`readily be moved, examined, and sorted in single file, i.e.,
`one at a time.
`
`0165
`
`(cited in Paper 21, Patent Owner's Response, pp. 33-34).
`
`Ex. 1004 (Link), 77 164-165
`
`

`
`Link 1] 173 Does Not Disclose Same Constant Pressure
`
`[0173] The microfluidic device of the present invention
`includes one or more inlet modules. An “inlet module” is an
`area of a microfluidic substrate device that receives mol-
`
`ecules, cells, small molecules or particles for additional
`coalescence, detection and/or sorting. The inlet module can
`contain one or more inlet channels, wells or reservoirs,
`openings, and other features which facilitate the entry of
`molecules, cells, small molecules or particles into the sub-
`strate. A substrate may contain more than one inlet module
`if desired. Different sample inlet channels can communicate
`with the main channel at different inlet modules. Alternately,
`different sample inlet channels can communication with the
`main channel at the same inlet module. The inlet module is
`in fluid communication with the main channel. The inlet
`
`module generally comprises a junction between the sample
`inlet channel and the main channel such that a solution of a
`
`sample (ie, a fluid containing a sample such as molecules,
`cells, small molecules (organic or inorganic) or particles) is
`introduced to the main charmel and forms a plurality of
`droplets. The sample solution can be pressurized. The
`sample inlet channel can intersect the main channel such that
`the sample solution is introduced into the main channel at an
`angle perpendicular to a stream of fluid passing through the
`main channel. For example, the sample inlet channel and
`main charmel intercept at a T-shaped junction; i.e., such that
`the sample inlet channel is perpendicular (90 degrees) to the
`main channel. However, the sample inlet channel can inter-
`cept the main channel at any angle, and need not introduce
`the sample fluid to the main channel at an angle that is
`perpendicular to that flow. The angle between intersecting
`channels is in the range of from about 60 to about 120
`degrees. Particular exemplary angles are 45, 60, 90, and 120
`degrees.
`
`Ex. 1004 (Link), fl 173
`
`(cited in Paper 21, Patent Owner's Response, p. 34).
`
`

`
`Link 1] 139 Does Not Disclose Same Constant Pressure
`
`[0139] The present invention also provides compositions
`and methods for creating emulsion of the sample fluid (e.g.
`droplets) prior to the introduction of the sample fluid into the
`microfluidic devices of the present invention. More specifi-
`cally, the methods are directed to the creating sample droplet
`emulsions “ofi” chip”, for example in a syringe. In order to
`create a monodisperse emulsion directly from a library well,
`a nozzle is formed directly into the fitting used to connect the
`collection syringe to a syringe tip (e.g. capillary tubing), as
`shown in FIG. 8.
`
`
`
`Ex. 1004 (Link), 7] 139
`
`
`
`Aspiration of the sample can be accom-
`plished by running the collection syringe in withdrawal
`mode at a flow rate (Q3) above the flow rate of the two oil
`syringes (Step 1 in FIG. 8). The difference in flow would
`correspond to the flow rate aspirated from the sample well.
`When the appropriate volume of sample has been loaded
`into the capillary tubing,
`the capillary tubing would be
`removed from the sample well, an air bubble, and possibly
`a cleaning solution would be aspirated (Step 2 in FIG. 8).
`When almost all of the sample has been emulsified, the
`collection syringe withdrawal rate would either be reduced
`below the oil flow rates, stopped, or set to infiise at some
`nominal rate (Step 3 in FIG. 8). The remaining sample, air,
`cleaning solution, etc, left in the capillary would be flushed
`back out into a cleaning well and the outside of the capillary
`would be cleaned at the “wash station.” When the capillary
`is completely clean, the process would repeat for the next
`library element.
`
`
`
`(cited in Paper 21, Patent Owner's Response, p. 22).
`
`

`
`Link 1] 173-74 Do Not Disclose Same Constant Pressure
`
`[0174] Embodiments of the invention are also provided in
`which there are two or more inlet modules introducing
`droplets of samples into the main channel. For example, a
`first inlet module may introduce droplets of a first sample
`into a flow of fluid in the main channel and a second inlet
`
`module may introduce droplets of a second sample into the
`flow of fluid in main channel, and so forth. The second inlet
`module is preferably downstream from the first inlet module
`(eg., about 30 pm). The fluids introduced into the two or
`more different inlet modules can comprise the same fluid or
`the same type of fluid (e.g., different aqueous solutions). For
`example, droplets of an aqueous solution containing an
`enzyme are introduced into the main channel at the first inlet
`module and droplets of aqueous solution containing a sub-
`strate for the enzyme are introduced into the main channel
`at the second inlet module. Alternatively, the droplets intro-
`duced at
`the different inlet modules may be droplets of
`different fluids which may be compatible or incompatible.
`For example, the diflerent droplets may be difierent aqueous
`solutions, or droplets introduced at a first inlet module may
`be droplets of one fluid (e.g., an aqueous solution) whereas
`droplets introduced at a second inlet module may be another
`fluid (e.g., alcohol or oil).
`
`[0173] The microfluidic device of the present invention
`includes one or more inlet modules. An “inlet module” is an
`area of a microfluidic substrate device that receives mol-
`
`ecules, cells, small molecules or particles for additional
`coalescence, detection and/or sorting. The inlet module can
`contain one or more inlet channels, wells or reservoirs,
`openings, and other