Office Action 11: Non-Final Rejection (2023-06-01)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
Amendments to the claims:

Please amend the claims as follows.

1\. (Currently Amended) A method for droplet formation for sample preparation for sequencing, the method comprising:
providing a first aqueous fluid from a first reservoir into a first inlet channel on a microfluidic substrate, the first aqueous fluid comprising biological material;
providing, via a second inlet channel, a second aqueous fluid comprising primers;
coalescing the first aqueous fluid with the second aqueous fluid to form an aqueous sample fluid;
providing an immiscible carrier fluid via at least one oil supply line;
applying a pressure to flow the immiscible carrier fluid into a nozzle disposed within the immiscible carrier fluid and into a main channel within the nozzl, , ; and
adjusting pressure in one or more of the inlet channels, thereby to produce a plurality of droplets comprising the aqueous sample fluid in the main channel wherein the nozzle comprises a ferrule immersed, by design, within the immiscible carrier fluid, thereby inhibiting \[\[s\]\] the aqueous sample fluid from wetting surfaces of the nozzle or the main channel.

2\. (Cancelled)

3\. (Currently Amended) The method of claim 1 \[\[2\]\], wherein surfaces of the nozzle and ferrule are wetted entirely by the carrier fluid.

4\. (Previously Presented) The method of claim 1, wherein the nozzle has complete isolation from any continuous aqueous phase.

5-11. (Cancelled) 610

12\. (Previously Presented) The method of claim 1, wherein the aqueous fluids further comprise reagents for conducting an amplification reaction.

13\. (Previously Presented) The method of claim 12, wherein the amplification reaction is a PCR reaction and the method comprises performing the PCR reaction in the one of the droplets to yield PCR products and breaking the droplets to release the PCR products from the droplets.

14-20. (Cancelled) 1519

21\. (Previously Presented) The method of claim I, wherein the oil supply line provides a constant flow of the carrier fluid into the nozzle while the plurality of droplets are formed.

22-30. (Cancelled) 2329

31\. (Previously Presented) The method of claim I, wherein the immiscible carrier fluid comprises an oil.

32\. (Previously Presented) The method of claim 3 I, wherein the oil comprises a fluorocarbon oil.

33\. (Previously Presented) The method of claim 3 1, wherein the oil comprises a surfactant.

34\. (Previously Presented) The method of claim 33, wherein the surfactant is a fluorosurfactant.

35-38. (Cancelled) 3637

39\. (New) The method of claim 13, wherein the microfluidic substrate is a chip that includes heating and cooling lines.

40\. (New) The method of claim 39, wherein the PCR reaction is performed by sending the one of the droplets through a serpentine path between the heating and cooling lines in the chip.
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OFFICE ACTION:
DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection.  Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.  Applicant's submission filed on January 31, 2023 has been entered.
Preliminary Remark
Claims 2, 5-11, 14-20, 22-30, and 35-38 are canceled.
Claims 39 and 40 are new.
Information Disclosure Statement
The IDS received on October 28, 2022 is proper and is being considered by the Examiner.
Claim Rejections - 35 USC § 112
The rejection of claims 1-4, 12, 13, 21, 23, and 31-34 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter, made in the Office Action mailed on October 31, 2022 is withdrawn in view of the Amendment received on January 31, 2023.
The new matter rejection of claims 1-4, 12, 13, 21, 23, and 31-34 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement., made in the Office Action mailed on October 31, 2022 is withdrawn in view of the Amendment received on January 31, 2023.
Rejection – New Grounds, Necessitated by Amendment
The following is a quotation of 35 U.S.C. 112(b):
(b)  CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 3 and 21 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA  35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 is indefinite because the claim 3 does not appear to further limit the parent claim for the following reasons.
The parent claim, claim 1 has been amended to require that the method employs a nozzle that comprises a ferrule “immersed, by design, within the immiscible carrier fluid sample, thereby inhibiting the aqueous sample fluid from wetting the surfaces of the nozzle or the main channel”.
Claim 3, depending from claim 1, recites that the “surfaces of the nozzle and ferrule are wetted entirely by the carrier fluid.”
Claim 3 no longer further limit its parent claim because the nozzle already comprises a ferrule which is “immersed” in the immiscible fluid.  By immersion, the surfaces of the nozzle and ferrule are wetted entirely by the immiscible fluid.  Therefore, there doesn’t appear to be any further limitation in claim 3.
Claim 21 recites the phrase, “the oil supply line”.  It is unclear which of the, “at least one oil supply line” the oil supply line is referring to.
Amending the phrase to recite, “the at least one oil supply line” would overcome this rejection.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 4, 12, 13, 21, 31, 39, and 40 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 584 586 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004) and Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49).
With regard to claim 1, Cho et al. teach a method of performing nucleic acid amplification reaction (“a method and apparatus for amplifying nucleic acids”, Abstract), the method comprising:
providing a first aqueous fluid from a first reservoir into a first inlet channel on a microfluidic substrate, the first aqueous fluid comprising a biological material (see Fig. 6, Target DNA reservoir 442);
providing via a second inlet channel, a second aqueous fluid comprising primer (see Fig. 6, see primer reservoir 444);
coalescing the first aqueous fluid with the second aqueous fluid to form an aqueous sample fluid (see Fig. 6, channel where the aqueous droplets merge therein prior to entry into main channel);
providing an immiscible carrier fluid via at least one oil supply line (see Fig. 6, see oil 152),
applying a pressure to flow the immiscible fluid into a main channel (“oil 131 … inserted into the reaction channel 130 via the respective inlet channels … 151 at a constant flow rate”, section [0053] evidences the presence of a source of pressure driven flow).
With regard to claims 12 and 13 the aqueous fluids further comprise reagents for conducting an amplification (see Fig. 6, element 446, “for example, polymerase, dNTPs, and buffer”, section [0076]).
With regard to claim 21, the oil flow is constant flow (see section [0053]).
With regard to claim 31, the immiscible fluid is oil (“oil introduction”, section [0076]).
With regard to claims 39 and 40, the microfluidic substrate is a chip that includes a heating and cooling lines (see Figure 7, T1, T2, and T3, wherein the PCR reaction is performed as the droplets travel past the different temperature zones of heating and cooling (also, “substrate 110 has two areas having different temperatures, for example, 65oC area and 95oC area … heating unit 120 may also be comprised of three or more heaters having different temperatures”, section [0050]).
Cho et al. do not disclose that the device comprises a nozzle disposed within the immiscible carrier fluid which leads into the main channel where emulsion droplets enter for nucleic acid amplification reaction, wherein the nozzle comprises a ferrule immersed in oil thereby inhibiting the aqueous droplets to wet the surfaces of the nozzle or the main channel (claims 1 and 3), wherein the nozzle has complete isolation from any continuous aqueous phase (claim 4).
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Kumacheva et al. teach a device for producing droplets within a channel, wherein in an embodiment, aqueous droplets are generated and enters a main channel that is prefilled with immiscible fluid, so as to prevent the aqueous droplets from contacting the main channel and its entry (see below Figure 21b, reproduced below):
“present invention discloses a versatile strategy of synthesis of polymeric particles using a ‘lab on chip’ with pre-designed size shape, morphology and composition” (page 9, lines 18-30)
“Figures 21a and 21b is a schematic illustration of the formulation of droplets by two different mechanism using the microfluidic reactor 120 … Immiscible liquids L1 and L2 (e.g., oil and aqueous phases) are forced into the narrow orifice …” (page 20, lines 23-26)
Bhagat et al. teach a prior art known means of coupling a channel to another channel via a ferrule (see Figure 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Cho et al. with the teachings of Kumacheva et al. and Benett et al., thereby arriving at the invention as claimed.
As discussed above, Cho et al. already teach a microfluidic device comprising separate reservoirs that provide template nucleic acids and reagents for amplification reaction, which are connected to a main channel flowing with oil.  The resulting configuration produces individual droplets of amplification reaction reagents that are surrounded by flowing oil which are flowed past different sections of the microfluidic chip zones having different temperatures.
While Cho et al. did not explicitly teach that the single channel which coalesces the template nucleic acid and amplification reagents leads into the main channel is completely surrounded by oil, such a configuration has been taught by Kumacheva et al. as shown above that result in the same result of providing an emulsified droplet of precisely controlled size (see page 9, lines 29-35).
As to connecting the main channel to the portion where the aqueous droplets are dispersed (see below left region where droplets are formed and the right region (b) the main channel where emulsion droplets flow through in the figure from
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Kumacheva et al.), use of any such prior known coupling means, such as ferrule would have been an obvious implementation, who use does not provide any perceivable functional advantage over that of Kumacheva et al..
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is prima facie obvious over the cited references.
Claims 32-34 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 584 586 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004) and Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49) as applied to claims 1, 3, 4, 12, 13, 21, 31, 39, and 40 above, and further in view of Dorfman et al. (Analytical Chemistry, 2005, vol. 77, pages 3700-3704).
The teachings of Cho et al., Kumacheva et al. and Bhagat et al. have been discussed above.
Cho et al., Kumacheva et al. and Bhagat et al. do not teach that the immiscible fluid oil should be fluorinated (claim 32) or comprise a fluorosurfactant (claims 33 and 34).
Dorfman et al., much like Cho et al., disclose a method of performing a continuous PCR reaction in aqueous droplets surrounded by immiscible oil (“[p]erforming continuous flow PCR for high-throughput clinical and quantitative applications require encapsulating the sample and preventing contamination between them … this can be accomplished in two ways: either the sample can be a droplet entrained in an immiscible solvent …”, page 3700, 2nd column).
Dorfman et al. disclose the use of fluorinated oil as the immiscible medium when performing the continuous emulsion PCR (“an improvement system for performing continuous flow PCR using droplets in an immiscible fluorinated solvent …”, page 3701, 1st column, 2nd paragraph; “primed with either the bulk fluorinated oil FC-40”, page 3701, 1st column, 3rd paragraph) or oil comprising a fluorosurfactant (“or FC-40 containing various amounts of fluoro alcohol surfactant”, page 3701, 1st column, 3rd paragraph).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Cho et al., Kumacheva et al. and Bhagat et al. with the teachings of Dorfman et al., thereby arriving at the invention as claimed for the following reasons.
Dorfman et al. discuss potential problems associated with performing emulsion droplet PCR in an immiscible medium:
“wall treatment [of the channel] are necessary to prevent adsorption of DNA to the walls and subsequent transfer between samples … wall contamination problem is a significant drawback … bubbles in the stream may introduce irregularities in the thermal history of the droplets, making the technique less attractive … flowing droplet system does not suffer from either of these drawbacks … However, the initial work on this system suffered from contamination between droplets” (page 3701, 1st column, 1st paragraph)
In solving this drawback, Dorfman et al. disclose that immiscible solvent, “prevent[ed] the droplet breakage … thereby eliminating the contamination between droplets” (page 3701, 1st column, 2nd paragraph), producing “adequately reproducible amplification between different drops without any detectable contamination between them” (page 3701, 1st column, 2nd paragraph).
Based on such findings, one of ordinary skill in the art would have been motivated to utilize a fluorinated immiscible oil or oil comprising fluorosurfactants into the teachings of Cho et al., Kumacheva et al. and Bhagat et al., for the same recognized benefit of producing reproducible amplification reaction between emulsion droplets as well as preventing contamination therebetween.
Given that Cho et al., in combination with Kumacheva et al. performed emulsion amplification reaction in a flowing oil medium, said one of ordinary skill in the art would have also expected that the addition of fluorosurfactants or using fluorinated oil in the method of Cho et al./Kumacheva et al. would have yielded the same predictable benefit as observed by Dorfman et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries

APPLICANT REBUTTAL: Remarks
With entry of this amendment, claims 1, 4, 12, 13, 21, 31-34, 39, and 40 are pending, with claim 1 being the only independent claim. Claims 1 is amended. Support for the amendments may be found in the original application at least at lines 5-23 on page 29 and lines 1-10 on page 86.
No new matter is added.
Supplemental Amendment
The Office may enter a supplemental reply as provided for by 37 CFR 1.111, which gives examples of permissible amendments in an open list. MPEP 714.03(A) provides that, "This list is not exhaustive. The examiner has the discretion to approve the entry of a supplemental reply that is not listed above."
Applicant hereby asks the Examiner to enter this amendment. For completeness of the
written record, and to make every effort, in good faith, to advance prosecution of this application to allowance, Applicant offers the following remarks given below to address those grounds of rejection that were set forth in the previous Office Action that was mailed on June 1, 2023.
Rejections under 35 U.S.C.
Q
The Office Action on page 3 stated that claims 3 and 21 are rejected under 35 U.S.C. 112 (pre-AIA), second paragraph, as indefinite.
Claim 3 was cancelled, rendering the rejection as to that claim moot. Claim 21 was
amended for consistency with base claim 1. Applicant submits that the rejections under § 11 2 2 have been overcome.
Rejections under 35 U.S.C.
Q
The Office Action, at page 4, stated that claim 1, 3, 4, 12, 13, 21, 31, 39, and 40 are
rejected under §103 over EP 1574586A2 to Cho in view of WO 2005/103106 to Kumacheva and Bhagat, 2007, Re-usable quick-release interconnect for characterization of microfluidic systems, J Micromech Microeng 17:42-39. At page 9, claims 32-34 are so rejected over Cho, Kumacheva, and Bhagat and further in view of Dorfman, 2005, Anal Chem 77:3700-3704.
Applicant submits that the claims of this supplemental amendment are not obvious.
A determination of obviousness requires basic factual inquiries into the scope and content of the prior art as well as the differences between the claimed invention and the prior art. Graham v. John Deere Co., 383 U.S. 1 (1966). When a patent claim includes a limitation that is wholly missing from the prior art, then that prior art does not support a conclusion of obviousness. Arendi S.A.R.L. v. Apple, Inc., 832 F.3d 1355, 1366-67 (Fed. Cir. 2016).
Independent claim 1 recites a method for forming droplets of an aqueous sample fluid. The method includes flowing an immiscible carrier fluid into a nozzle that is immersed within the immiscible carrier fluid and into a main channel within the nozzle and adjusting pressure of the aqueous sample fluid in an inlet channel to produce a plurality of droplets of the aqueous sample fluid. Claim 1-as amended-recites that the nozzle is immersed, by design, within the immiscible carrier fluid, thereby preventing the aqueous sample fluid from wetting surfaces of the nozzle or the main channel.
The prior art does not teach or suggest a droplet formation nozzle that is immersed in oil and that therefore does not undergo any aqueous phase wetting.
Cho only reports injecting an aqueous solution of PCR reactants and a second fluid via different inlet channels into a channel to form aqueous droplets in which amplification is performed. EP 1574586 A2, [0015]. In Cho, amplification occurs in a reaction channel with a serpentine shape passing between two temperature areas. Id., [0045]. Cho never mentions any nozzle, much less any significance to the concept of phase wetting of surface, and thus does not teach or suggest a droplet-formation nozzle designed to avoid aqueous wetting.
Kumacheva reports producing polymer particles with pre-selected shapes by injecting a polymerizable fluid and an immiscible second fluid into a channel long enough that droplets have time to polymerize or harden into the intended shapes. WO 2005/103106, Abstract. Kumacheva shows an example in which silicone oil was inserted in a central channel while an aqueous solution was supplied to the outer channels, in which when the two liquids were forced through an orifice, a thread of silicone oil broke up into oil droplets dispersed within the aqueous continuous phase. Id., page 30, lines 16-21. Kumacheva does not teach or suggest any mechanism by which to form aqueous droplets surrounded by an immiscible carrier fluid. In fact, because Kumacheva forms droplets of silicone
oil surrounded by aqueous solution,
Kumacheva teaches and requires that the droplet formation mechanism be wetted by the aqueous phase, which teaches away from the subject matter of claim 1.
Bhagat only reports re-usable quick-release compression-based fluidic connections for microfluidic systems based on using O-rings to form a compression seal against a surface of the microfluidic device and around the inlet/outlet tubing, thus establishing connections to the macroworld and preventing any leaks at the ports. Bhagat 2007, Abstract. There is no mention of droplets or emulsions or immiscible liquids. Lacking any disclosure of immiscible liquids, Bhagat does not teach or suggest any droplet formation nozzle designed to avoid aqueous wetting.
Dorfman reports PCR in droplets flowing in an immiscible solvent. Dorfman, Abstract. Dorfman uses droplets in the immiscible solvent flowing in Teflon capillaries through zones of a compact heating system. Id., 3701. Dorfman reports aspirating droplets from a fluid reservoir into a capillary tube. Id., 3702. Dorfman explores variability in PCR results among droplets and observes that, even when the tip of the aspiration capillary is wetted with oil/surfactant mixture prior to aspiration, "variability in the amount of wetting fluorocarbon mixture of the tip" leads to different PCR droplet sizes. Id., 3703. Dorfman does not teach or suggest fully wetting a nozzle with an oil phase by having the nozzle immersed in the oil phase as a mechanism by which to avoid aqueous wetting of the nozzle. In fact, Dorfman explicitly states that the aspiration capillary experiences "variability in the amount of wetting fluorocarbon mixture of the tip". Id [which means that some amount is wetted by the oil and that another amount
is wetted by the aqueous phase
and that those amounts vary].
The Office has not found a droplet formation nozzle with a design that prevents aqueous fluid from wetting surfaces of the nozzle or a channel therein.
Because that element of claim 1, as amended, is missing from the prior art, claim 1 is not obvious. Because the only independent claim is not obvious, none of the claims are obvious. Applicant submits that the rejections under § 103 are overcome.
Conclusion

NEXT OFFICE ACTION: DETAILED ACTION
The present Office Action is responsive to the Amendment received on September 1, 2023 and the supplemental amendment received on October 23, 2023 (herein, referred together as “Amendment” or “Response”).
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Preliminary Remark
Claims 2, 3, 5-11, 14-20, 22-30, and 35-38 are canceled.
Claim Rejections - 35 USC § 112
The rejection of claims 3 and 21 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter, made in the Office Action mailed on June 1, 2023 is withdrawn in view of the Amendment.
Claim Rejections - 35 USC § 103
The rejection of claim 3 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 584 586 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004) and Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), made in the Office Action mailed on June 1, 2023 is withdrawn in view of the Amendment, canceling the rejected claim.
The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
The rejection of claims 1, 4, 12, 13, 21, 31, 39, and 40 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 584 586 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004) and Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), made in the Office Action mailed on June 1, 2023 is maintained for the reasons of record.
Applicants’ arguments presented in the Amendment received in the Amendment have been considered but they have not been found persuasive for the reasons set forth in the, “Response to Arguments” section.
The Rejection:
With regard to claim 1, Cho et al. teach a method of performing nucleic acid amplification reaction (“a method and apparatus for amplifying nucleic acids”, Abstract), the method comprising:
providing a first aqueous fluid from a first reservoir into a first inlet channel on a microfluidic substrate, the first aqueous fluid comprising a biological material (see Fig. 6, Target DNA reservoir 442);
providing via a second inlet channel, a second aqueous fluid comprising primer (see Fig. 6, see primer reservoir 444);
coalescing the first aqueous fluid with the second aqueous fluid to form an aqueous sample fluid (see Fig. 6, channel where the aqueous droplets merge therein prior to entry into main channel);
providing an immiscible carrier fluid via at least one oil supply line (see Fig. 6, see oil 152),
applying a pressure to flow the immiscible fluid into a main channel (“oil 131 … inserted into the reaction channel 130 via the respective inlet channels … 151 at a constant flow rate”, section [0053] evidences the presence of a source of pressure driven flow).
With regard to claims 12 and 13 the aqueous fluids further comprise reagents for conducting an amplification (see Fig. 6, element 446, “for example, polymerase, dNTPs, and buffer”, section [0076]).
With regard to claim 21, the oil flow is constant flow (see section [0053]).
With regard to claim 31, the immiscible fluid is oil (“oil introduction”, section [0076]).
With regard to claims 39 and 40, the microfluidic substrate is a chip that includes a heating and cooling lines (see Figure 7, T1, T2, and T3, wherein the PCR reaction is performed as the droplets travel past the different temperature zones of heating and cooling (also, “substrate 110 has two areas having different temperatures, for example, 65oC area and 95oC area … heating unit 120 may also be comprised of three or more heaters having different temperatures”, section [0050]).
Cho et al. do not disclose that the device comprises a nozzle disposed within the immiscible carrier fluid which leads into the main channel where emulsion droplets enter for nucleic acid amplification reaction, wherein the nozzle comprises a ferrule immersed in oil thereby inhibiting the aqueous droplets to wet the surfaces of the nozzle or the main channel (claims 1 and 3), wherein the nozzle has complete isolation from any continuous aqueous phase (claim 4).
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Kumacheva et al. teach a device for producing droplets within a channel, wherein in an embodiment, aqueous droplets are generated and enters a main channel that is prefilled with immiscible fluid, so as to prevent the aqueous droplets from contacting the main channel and its entry (see below Figure 21b, reproduced below):
“present invention discloses a versatile strategy of synthesis of polymeric particles using a ‘lab on chip’ with pre-designed size shape, morphology and composition” (page 9, lines 18-30)
“Figures 21a and 21b is a schematic illustration of the formulation of droplets by two different mechanism using the microfluidic reactor 120 … Immiscible liquids L1 and L2 (e.g., oil and aqueous phases) are forced into the narrow orifice …” (page 20, lines 23-26)
Bhagat et al. teach a prior art known means of coupling a channel to another channel via a ferrule (see Figure 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Cho et al. with the teachings of Kumacheva et al. and Benett et al., thereby arriving at the invention as claimed.
As discussed above, Cho et al. already teach a microfluidic device comprising separate reservoirs that provide template nucleic acids and reagents for amplification reaction, which are connected to a main channel flowing with oil.  The resulting configuration produces individual droplets of amplification reaction reagents that are surrounded by flowing oil which are flowed past different sections of the microfluidic chip zones having different temperatures.
While Cho et al. did not explicitly teach that the single channel which coalesces the template nucleic acid and amplification reagents leads into the main channel is completely surrounded by oil, such a configuration has been taught by Kumacheva et al. as shown above that result in the same result of providing an emulsified droplet of precisely controlled size (see page 9, lines 29-35).
As to connecting the main channel to the portion where the aqueous droplets are dispersed (see below left region where droplets are formed and the right region (b) the main channel where emulsion droplets flow through in the figure from
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Kumacheva et al.), use of any such prior known coupling means, such as ferrule would have been an obvious implementation, who use does not provide any perceivable functional advantage over that of Kumacheva et al..
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is prima facie obvious over the cited references.
Response to Arguments:
Applicants traverse the rejection.
Applicants contend that claim 1 as amended recites that the nozzle is immersed by design, within the immiscible carrier fluid thereby preventing the aqueous fluid from wetting the surface of the nozzle or the main channel (page 6, Response).
Applicants contend that Cho (of record), while disclosing generation of aqueous droplets (PCR droplets) within immiscible carrier fluid (oil), the artisan does not teach or suggest the concept of a nozzle or the concept of phase wetting of surface (page 6, Response)
With regard to Kumacheva’s teachings, Applicants are completely silent on whether the artisans teach the concept of using a nozzle.  Rather, Applicants stress that the carrier fluid employed by Kumacheva is aqueous solution, and the droplets formed therein are oil droplets.  By Applicants’ silence, the Office assumes that Applicants are admitting that Kumacheva does teach a droplet generation device which comprises a nozzle, and Applicants’ contention is that because the carrier fluid employed by Kumacheva is aqueous, nozzle is “wetted” by the aqueous phase.
This argument has been carefully considered but have not been found persuasive because the rejection is based on a combination of references.
As admitted by Applicants, Cho teaches the generation of aqueous droplets surrounded by an immiscible carrier fluid (i.e., oil) for the purposes of performing a PCR reaction, well-known in the art as emulsion PCR.  The fundamental basis of this technology rests on the immiscible nature of the two fluids, which are oil and water (or aqueous).  The two are immiscible and therefore, one forms a droplet inside the body of another.
Whether the oil forms a droplet inside the body of an aqueous fluid, or the aqueous fluid forms a droplet inside the body of oil, is to be expected, such properties would have been recognized by one of ordinary skill in the art.
While Applicants attempt to make much of Kumacheva generating droplets of oil in the body of an aqueous carrier fluid, one of ordinary skill in the art would have recognized that the droplet formations would occur in the reverse orientation (i.e., aqueous in the body of oil carrier) because the two, as discussed above, are recognized as being immiscible.
Because the rejection is based on Cho in view of Kumacheva (not the reverse), one of ordinary skill in the art who is considering the teachings of Cho, which involves the generation of aqueous droplets containing PCR reagents in the immiscible, oil carrier fluid, when viewing the teachings of Kumacheva who teach the use of a nozzle configuration in which oil droplets are generated, would have recognized that the same nozzle configuration would have generated the aqueous droplets in oil carrier.
Applicants are reminded that when considering obviousness, the references being combined need not be wholly combined in their entirety.
“Additionally, Patent Owner’s argument that the Petition fails to explain why a POSITA “would combine particular components from the prior art in a specific way” seeks to impose a bodily incorporation requirement that is not required for obviousness. Prelim. Resp. 24. “The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference,” but rather whether “a skilled artisan would have been motivated to combine the teachings of the prior art references to achieve the claimed invention.” Allied Erecting & Dismantling Co. v. Genesis Attachments, LLC, 825 F.3d
1373, 1381 (Fed. Cir. 2016) (internal citations omitted).
Also, in KSR International Co v. Teleflex Inc, the Supreme Court stated that, “A person of ordinary skill in the art is also a person of ordinary creativity, not an automation” (82 USPQ2d at 1397) and that “in many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle” and take into account, “the inference and creative steps that a person of ordinary skill in the art would employ” (82 USPQ2d at 1396).
Lastly, the Office notes that the droplet generation means taught by Kumacheva satisfies the “nozzle” construct:
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As seen, L1 channels are carrying a carrier fluid, while L2 is an immiscible fluid (to L1) which become droplets in the L1 fluid body.  The opening entrance through which L2 fluid travel through to become droplets is considered a nozzle, and this nozzle structure is surrounded at all times by L1 carrier fluid and provides an opening into the main channel (the channel located right side of the nozzle where droplets are generated), and where formed droplets are completely surrounded the carrier fluid.
Kiss et al. (Analytical Chemistry, 2008, vol. 80, pages 8975-8981) evidence that the Office’s interpretation of the term, “nozzle” is consistent with the art, wherein the artisans, refer to a structure found in Fig 1B (see below), wherein the opening through which aqueous fluid pass through into a main channel (forming droplets) is referred to as a nozzle.
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“(B) Optical image of droplet generation at the nozzle” (see Figure 1 description)
Therefore, Applicants’ arguments are not found persuasive and the rejection is maintained.
The rejection of claims 32-34 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 584 586 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004) and Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49) as applied to claims 1, 4, 12, 13, 21, 31, 39, and 40 above, and further in view of Dorfman et al. (Analytical Chemistry, 2005, vol. 77, pages 3700-3704), made in the Office Action mailed on June 1, 2023 is maintained for the reasons of record.
Applicants do not present any new arguments in the Amendment, but rely solely on their arguments presented for the rejection over Cho et al., Kumacheva et al. and Bhagat et al. which have been fully responded to above.
Because no additional arguments are presented for the instant rejection, the rejection is maintained for the reasons already of record.
The Rejection:
The teachings of Cho et al., Kumacheva et al. and Bhagat et al. have been discussed above.
Cho et al., Kumacheva et al. and Bhagat et al. do not teach that the immiscible fluid oil should be fluorinated (claim 32) or comprise a fluorosurfactant (claims 33 and 34).
Dorfman et al., much like Cho et al., disclose a method of performing a continuous PCR reaction in aqueous droplets surrounded by immiscible oil (“[p]erforming continuous flow PCR for high-throughput clinical and quantitative applications require encapsulating the sample and preventing contamination between them … this can be accomplished in two ways: either the sample can be a droplet entrained in an immiscible solvent …”, page 3700, 2nd column).
Dorfman et al. disclose the use of fluorinated oil as the immiscible medium when performing the continuous emulsion PCR (“an improvement system for performing continuous flow PCR using droplets in an immiscible fluorinated solvent …”, page 3701, 1st column, 2nd paragraph; “primed with either the bulk fluorinated oil FC-40”, page 3701, 1st column, 3rd paragraph) or oil comprising a fluorosurfactant (“or FC-40 containing various amounts of fluoro alcohol surfactant”, page 3701, 1st column, 3rd paragraph).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to combine the teachings of Cho et al., Kumacheva et al. and Bhagat et al. with the teachings of Dorfman et al., thereby arriving at the invention as claimed for the following reasons.
Dorfman et al. discuss potential problems associated with performing emulsion droplet PCR in an immiscible medium:
“wall treatment [of the channel] are necessary to prevent adsorption of DNA to the walls and subsequent transfer between samples … wall contamination problem is a significant drawback … bubbles in the stream may introduce irregularities in the thermal history of the droplets, making the technique less attractive … flowing droplet system does not suffer from either of these drawbacks … However, the initial work on this system suffered from contamination between droplets” (page 3701, 1st column, 1st paragraph)
In solving this drawback, Dorfman et al. disclose that immiscible solvent, “prevent[ed] the droplet breakage … thereby eliminating the contamination between droplets” (page 3701, 1st column, 2nd paragraph), producing “adequately reproducible amplification between different drops without any detectable contamination between them” (page 3701, 1st column, 2nd paragraph).
Based on such findings, one of ordinary skill in the art would have been motivated to utilize a fluorinated immiscible oil or oil comprising fluorosurfactants into the teachings of Cho et al., Kumacheva et al. and Bhagat et al., for the same recognized benefit of producing reproducible amplification reaction between emulsion droplets as well as preventing contamination therebetween.
Given that Cho et al., in combination with Kumacheva et al. performed emulsion amplification reaction in a flowing oil medium, said one of ordinary skill in the art would have also expected that the addition of fluorosurfactants or using fluorinated oil in the method of Cho et al./Kumacheva et al. would have yielded the same predictable benefit as observed by Dorfman et al.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id.  at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL.  Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action.  In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action.  In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Inquiries

CITED REFERENCE SUMMARIES: Ref EP1574586A2: Claims:
A method for amplifying nucleic acids, which comprises: introducing into a reaction vessel via different inlet channels a reactant
aqueous solution containing reactants for nucleic acid amplification and a fluid that is
phase-separated from the reactant aqueous solution and does not participate in
amplification reaction; creating a plurality of reactant aqueous solution droplets surrounded by the
fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel;
and amplifying the nucleic acids in the reactant aqueous solution droplets.
introducing into a reaction vessel via different inlet channels a reactant
aqueous solution containing reactants for nucleic acid amplification and a fluid that is
phase-separated from the reactant aqueous solution and does not participate in
amplification reaction; creating a plurality of reactant aqueous solution droplets surrounded by the
fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel;
and amplifying the nucleic acids in the reactant aqueous solution droplets.
introducing into a reaction vessel via different inlet channels a reactant
aqueous solution containing reactants for nucleic acid amplification and a fluid that is
phase-separated from the reactant aqueous solution and does not participate in
amplification reaction;
creating a plurality of reactant aqueous solution droplets surrounded by the
fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel;
and
amplifying the nucleic acids in the reactant aqueous solution droplets.
The method of claim 1, wherein the fluid is at least one selected from
the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and
vegetable oil.
The method of claim 1, wherein the operation of amplifying the
nucleic acids is performed by one selected from the group consisting of polymerase
chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification
(SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated
amplification (TMA), and loop-mediated isothermal amplification (LAMP).
The method of claim 3, wherein the operation of amplifying the
nucleic acids is performed by PCR.
The method of claim 4, wherein the operation of amplifying the
nucleic acids is performed by PCR in which the entire surface of a substrate is
repeatedly heated and cooled to a predetermined temperature.
The method of claim 4, wherein the operation of amplifying the
nucleic acids is performed by continuous-flow PCR in which a substrate is heated to
have at least two different temperature areas and the reactant aqueous solution
droplets alternately and repeatedly pass through the different temperature areas.
The method of claim 6, wherein the substrate is heated to have at 
least two different temperature areas using a plurality of independently controllable
heaters.
The method of claim 4, wherein the operation of introducing the
reactant aqueous solution and the fluid into the reaction vessel further comprises
introducing into the reaction vessel at least one aqueous solution of a negative
control and a positive control for quantitative assay and a standard sample aqueous
solution via respective inlet channels different from the inlet channels for the
introduction of the reactant aqueous solution and the fluid, and    the operation of creating the reactant aqueous solution droplets further
comprises creating a plurality of control aqueous solution droplets and a plurality of
standard sample aqueous solution droplets by contacting the at least one aqueous
solution of the negative control and the positive control and the standard sample
aqueous solution with the fluid in the reaction vessel.
The method of claim 8, wherein in the operation of introducing the
reactant aqueous solution and the fluid into the reaction vessel, the standard sample
aqueous solution is adjusted to at least two concentrations and then introduced into
the reaction vessel.
The method of claim 4, wherein in the operation of introducing the
reactant aqueous solution and the fluid into the reaction vessel, at least two reactant
aqueous solutions containing at least two primers are introduced into the reaction
vessel via respective inlet channels.
The method of claim 4, wherein in the operation of introducing the
reactant aqueous solution and the fluid into the reaction vessel, a nucleic acid
aqueous solution, a common component containing polymerase and dNTP, and a
primer are introduced into respective inlet channels and mixed before being
introduced into the reaction vessel to form the reactant aqueous solution.
The method of claim 1, wherein the reaction vessel comprises two
primary reaction channels and a single secondary reaction channel connected to a
terminal end of each of the two primary reaction channels, and the operation of 
introducing the reactant aqueous solution and the fluid into the reaction vessel, the
operation of creating the reactant aqueous solution droplets, and the operation of
amplifying the nucleic acids are performed in each of the two primary reaction
channels, and    wherein after the operation of amplifying the nucleic acids, the method further
comprises: combining the reactant aqueous solution droplets in a starting end of the
secondary reaction channel to create a plurality of combined reactant aqueous
solution droplets in the secondary reaction channel; and amplifying the nucleic acids in the combined reactant aqueous solution
droplets.
combining the reactant aqueous solution droplets in a starting end of the
secondary reaction channel to create a plurality of combined reactant aqueous
solution droplets in the secondary reaction channel; and amplifying the nucleic acids in the combined reactant aqueous solution
droplets.
combining the reactant aqueous solution droplets in a starting end of the
secondary reaction channel to create a plurality of combined reactant aqueous
solution droplets in the secondary reaction channel; and
amplifying the nucleic acids in the combined reactant aqueous solution
droplets.
The method of claim 1, wherein the reaction vessel comprises at least
one reaction chamber, and    wherein after the operation of amplifying the nucleic acids in the reactant
aqueous solution droplets, the method further comprises: combining the reactant aqueous solution droplets in the reaction chamber;
and amplifying the nucleic acids in the combined reactant aqueous solution
droplets.
combining the reactant aqueous solution droplets in the reaction chamber;
and amplifying the nucleic acids in the combined reactant aqueous solution
droplets.
combining the reactant aqueous solution droplets in the reaction chamber;
and
The method of claim 13, wherein the operation of combining the
reactant aqueous solution droplets is carried out by centrifugation.
An apparatus for amplifying nucleic acids, which comprises: a substrate; a reaction vessel formed inside of the substrate; at least one first inlet channel formed inside the substrate, connected to an
end of the reaction vessel, and allowing introduction of a reactant aqueous solution
containing reactants for nucleic acid amplification into the reaction vessel; a second inlet channel formed inside the substrate, connected to the end of
the reaction vessel, and allowing introduction of a fluid that is phase-separated from
the reactant aqueous solution and does not participate in amplification reaction into
the reaction vessel; and a heating unit installed on the substrate and heating the substrate, the reactant aqueous solution contacting with the fluid to create a plurality of
reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and
the nucleic acids being amplified in the reactant aqueous solution droplets.
a substrate; a reaction vessel formed inside of the substrate; at least one first inlet channel formed inside the substrate, connected to an
end of the reaction vessel, and allowing introduction of a reactant aqueous solution
containing reactants for nucleic acid amplification into the reaction vessel; a second inlet channel formed inside the substrate, connected to the end of
the reaction vessel, and allowing introduction of a fluid that is phase-separated from
the reactant aqueous solution and does not participate in amplification reaction into
the reaction vessel; and a heating unit installed on the substrate and heating the substrate, the reactant aqueous solution contacting with the fluid to create a plurality of
reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and
the nucleic acids being amplified in the reactant aqueous solution droplets.
a reaction vessel formed inside of the substrate;
at least one first inlet channel formed inside the substrate, connected to an
end of the reaction vessel, and allowing introduction of a reactant aqueous solution
containing reactants for nucleic acid amplification into the reaction vessel;
a second inlet channel formed inside the substrate, connected to the end of
the reaction vessel, and allowing introduction of a fluid that is phase-separated from
the reactant aqueous solution and does not participate in amplification reaction into
the reaction vessel; and
a heating unit installed on the substrate and heating the substrate,
the reactant aqueous solution contacting with the fluid to create a plurality of
reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and
the nucleic acids being amplified in the reactant aqueous solution droplets.
The apparatus of claim 15, wherein the fluid is at least one selected
from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil,
and vegetable oil.
The apparatus of claim 15, wherein the substrate has a hydrophobic
surface property.
The apparatus of claim 17, wherein the substrate is made of one
material selected from the group consisting of poly(dimethylsiloxane) (PDMS), silicon,
silicon dioxide, plastic, and glass.
The apparatus of claim 15, wherein the substrate comprises a lower
substrate and an upper substrate, and the reaction vessel, the first inlet channel, and
the second inlet channel are formed between the lower substrate and the upper
substrate.
The apparatus of claim 15, wherein the nucleic acids are amplified in
the reactant aqueous solution droplets by one selected from the group consisting of
PCR, LCR, SDA, NASBA, TMA, and LAMP.
The apparatus of claim 20, wherein the nucleic acids are amplified by
PCR in the reactant aqueous solution droplets.
The apparatus of claim 21, wherein the reaction vessel is a
serpentine reaction channel, the first inlet channel and the second inlet channel are
connected to a staring end of the reaction channel, and an outlet port for releasing
the fluid and an amplification product is connected to a terminal end of the reaction
channel.
The apparatus of claim 22, wherein the heating unit is installed at a
lower surface of the substrate and comprises at least two heaters for heating the
substrate to allow the substrate to have at least two different temperature areas.
The apparatus of claim 22, wherein the heating unit comprises a
plurality of independently controllable heaters installed in parallel at a lower surface
of the substrate to heat the substrate so that the substrate has at least two
temperature areas.
The apparatus of claim 23, wherein the reaction channel alternately
and repeatedly passes through the temperature areas.
The apparatus of claim 22, wherein the second inlet channel linearly
extends from the starting end of the reaction channel and the first inlet channel is
formed perpendicularly to the second inlet channel.
The apparatus of claim 22, further comprising: a third inlet channel formed inside the substrate, connected to the end of the
reaction channel, and allowing introduction of at least one selected from a negative
control aqueous solution and a positive control aqueous solution for quantitative
assay into the reaction channel; and a fourth inlet channel formed inside the substrate, connected to the end of
the reaction channel, and allowing introduction of a standard sample aqueous
solution for quantitative assay into the reaction channel.
a third inlet channel formed inside the substrate, connected to the end of the
reaction channel, and allowing introduction of at least one selected from a negative
control aqueous solution and a positive control aqueous solution for quantitative
assay into the reaction channel; and a fourth inlet channel formed inside the substrate, connected to the end of
the reaction channel, and allowing introduction of a standard sample aqueous
solution for quantitative assay into the reaction channel.
a third inlet channel formed inside the substrate, connected to the end of the
reaction channel, and allowing introduction of at least one selected from a negative
control aqueous solution and a positive control aqueous solution for quantitative
assay into the reaction channel; and
a fourth inlet channel formed inside the substrate, connected to the end of
the reaction channel, and allowing introduction of a standard sample aqueous
solution for quantitative assay into the reaction channel.
The apparatus of claim 27, wherein the fourth inlet channel is
connected to at least two standard sample inlet channels for introduction of standard
samples and a distilled water inlet channel for diluting the standard samples to a
predetermined concentration.
The apparatus of claim 28, wherein the fourth inlet channel has a
serpentine shape.
The apparatus of claim 22, wherein the apparatus comprises at least 
two first inlet channels and the first inlet channels are correspondingly connected to
at least two primer inlet channels for introduction of at least two different primers into
the first inlet channels.
The apparatus of claim 30, wherein each of the first inlet channels
has a serpentine shape.
The apparatus of claim 22, wherein the first inlet channel is
connected to a primer inlet channel for introduction of a primer into the first inlet
channel and a common component inlet channel for introduction of a common
component comprising polymerase and dNTP into the first inlet channel.
The apparatus of claim 32, wherein a portion of a starting end side of
the reaction channel has a serpentine shape.
The apparatus of claim 15, wherein the reaction vessel comprises two
serpentine primary reaction channels and a single serpentine secondary reaction
channel connected to a terminal end of each of the two primary reaction channels,
and the first inlet channel and the second inlet channel are connected to starting
ends of the two primary reaction channels.
The apparatus of claim 34, wherein the heating unit comprises a
plurality of independently controllable heaters installed at a lower surface of the
substrate to provide two temperature areas for each of the two primary reaction
channels and the single secondary reaction channel.
The apparatus of claim 15, wherein the reaction vessel is a reaction
chamber, the first inlet channel and the second inlet channel are connected to an
end of the reaction chamber, and an outlet channel for releasing the fluid and an
amplification product is connected to the other end of the reaction chamber.
The apparatus of claim 36, wherein the apparatus comprises a
plurality of reaction chambers, first inlet channels, second inlet channels, and outlet
channels.
The apparatus of claim 36, wherein the heating unit comprises a
single heater installed at a lower surface of the substrate and heating the entire
surface of the substrate to a predetermined temperature.
Ref WO2005103106A1: Claims:
A process for producing polymer particles of predetermined size and/or shape, and /or morphology, comprising the steps of: a) injecting a first fluid comprising a constituent which can harden into a microfluidic channel; b) injecting at least a second fluid into the microfluidic channel for causing the first fluid to forms into fluidic droplets within the at least second fluid causing the fluidic droplets to flow through the microfluidic channel, the microfluidic channel being sufficiently long so that the fluidic droplets harden into particles of predetermined size and/or shape while flowing through the channel; and c) collecting the hardened particles of predetermined size and/or shape from the microfluidic, channel.
The process according to claim 1 , wherein the fluidic droplets harden by any one of ionic crosslinking, hydrogen bonding, chelation, complexation and combinations thereof.
The process according to claim 1 , wherein the first fluid comprises a polymerizable constituent, and wherein said fluidic droplets harden by polymerizing into polymer particles.
The process according to claims 1 , 2 or 3 wherein the microfluidic channel is constructed and configured to produce fluidic droplets having a pre-selected size and or shape.
The process according to claims 1 , 2, 3 or 4 wherein the microfluidic channel has a cross-sectional shape being any one of circular, oval, triangular, irregular, square, and rectangular.
The process according to claims 1 , 2, 3, 4 or 5 wherein the microfluidic channel has an aspect ratio (length to average cross sectional dimension) of at least about 10:1.
The process according to claims 1 , 2, 3, 4 or 6 wherein the first and second fluids are each injected with a controlled flow rate into the microfluidic channel.
The process according to claims 1 , 2, 3, 4, 5, 6 or 7 wherein a diameter of the fluidic droplets formed in step b) is controlled by controlling flow rates and flow rate ratios of the first and second fluids, controlling dimensions of said microfluidic channel, properties of said first and second fluids including viscosity and interfacial tension between the first and second fluids, controlling properties of the polymerizable constituent, and wherein said microfluidic channel includes a portion constructed with pre-selected cross-sectional shape and dimensions to give a desired shape to said fluidic droplets upon transit of said fluidic droplets through said portion of said fluidic channel.
The process according to any one of claims 1 to 8 wherein said fluidic droplets have a diameter d, and wherein said portion of the microfluidic channel is constructed with pre-selected cross-sectional shape and dimensions selected so that d is larger than at least one of the dimensions of the microfluidic channel so that fluidic droplets with non-spherical shapes are formed upon transit through said portion of the microfluidic channel with preselected cross-sectional shape and dimensions so that said collected polymer particles have a non-spherical shape.
The process according to claim 9 wherein an aspect ratio for said non- spherical droplets is varied by changing a ratio between fluidic droplet volume and said dimensions of the microfluidic channel.
The process according to any one of claims 1 to 8 wherein said fluidic droplets have a diameter d, and wherein said portion of the microfluidic channel has a width w and a height h, and wherein when said portion of the  microfluidic channel is constructed so that w > d and h > d, and a flow rate of the fluidic droplets through the microfluidic channel is selected to be slow enough so that the fluidic droplets maintain a spherical shape so that said collected polymer particles have a spherical shape.
The process according to ' any one of claims 1 to 8 wherein said fluidic droplets have a diameter d, and wherein said portion of the microfluidic channel has a width wand a height h, and wherein when said portion of the microfluidic channel is constructed so that w > d and h > d, and a flow rate of the fluidic droplets through the microfluidic channel is selected to be high enough so that the fluidic droplets acquire an ellipsoidal shape so that said collected polymer particles have an ellipso
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