Office Action 13: Non-Final Rejection (2024-05-23)
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, via at least one inlet channel on a microfluidic substrate,an aqueous sample fluid comprising nucleic acid and at least one primer ;
providing an immiscible firstcarrier fluid via at least one oil supply line;
applying a pressure to flow the immiscible firstcarrier fluid into a main channel on the microfluidic substrate;
adjusting pressure in the at least one inlet channel\[\[s\]\], thereby \[\[to\]\] producing \[\[e\]\] a plurality of droplets comprising the aqueous sample fluid in the main channel
copying the nucleic acid by extending the at least one primer; and
transferring the plurality of droplets from the first carrier fluid into a droplet-destabilizing second fluid, thereby removing the aqueous sample fluid from the plurality of droplets.

2-3. (Cancelled)

4\. (Currently Amended) The method of claim 1, wherein the first carrier fluid flows into the main channel via a nozzle that has complete isolation from any continuous aqueous phase.

5-11. (Cancelled)610
12\. (Currently Amended) The method of claim 1, wherein the aqueous sample fluid\[\[s\]\] further comprises reagents for conducting an amplification reaction.

13\. (Currently Amended) ) The method of claim 12, wherein the amplification reaction is a PCR reaction and the method comprises performing the PCR reaction .

14-20. (Cancelled)1519

21\. (Previously Presented) The method of claim 1, wherein the at least one 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 1, wherein the immiscible carrier fluid comprises an oil.

32\. (Previously Presented) The method of claim 31, wherein the oil comprises a fluorocarbon oil.

33\. (Previously Presented) The method of claim 31, 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\. (Previously Presented) The method of claim 13, wherein the microfluidic substrate is a chip that includes heating and cooling lines.
Application No. 15/480,739 40. (Currently Amended) The method of claim 39, wherein the PCR reaction is performed by sending the one droplet of the plurality droplets through a serpentine path between the heating and cooling lines in the chip.

41\. (New) The method of claim 1, wherein the droplet-destabilizing second fluid is an oil comprising a de-stabilizing surfactant.

42\. (New) The method of claim 41, further comprising centrifuging the plurality of droplets and the droplet-destabilizing second fluid to promote complete coalescence of the plurality of droplets.

43\. (New) The method of claim 41, wherein the de-stabilizing surfactant comprises a perfluorinated alcohol.

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
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, 2024 has been entered.
Preliminary Remark
Claims 2, 3, 5-11, 14-20, 22-30, and 35-38 are canceled.
Claims 41-43 are new.
Claim Objections – Necessitated by Amendment
Claim 41 is objected to because of the following informalities: claim 41 recites the words, “de-stabilizing.”  The hyphen should be removed.
Appropriate correction is required.
Claim Rejections - 35 USC § 112 – 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.
Claim 40 is 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 40 recites the phrase, “the one droplet of the plurality of droplets”.  It is unclear which of the many (plurality) of droplets, “the one droplet” is referring to.
Claim Rejections - 35 USC § 103
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 1 574 5861 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 October 31, 2023 is withdrawn in view of the Amendment received on January 31, 2024.
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 October 31, 2023 is withdrawn in view of the Amendment received on January 31, 2024.
Rejection – New Grounds, Necessitated by Amendment
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, 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 1 574 5862 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003).
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), and
copying the nucleic acid by extending the at least one primer (PCR copies the nucleic acid template).
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).
Cho et al. do not explicitly state that the emulsion droplets containing the amplification products are transferred from the oil carrier fluid into a droplet-destabilizing second fluid, and the aqueous sample fluid is removed from the plurality of droplets.
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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).
Leamon et al. teach a method of amplifying nucleic acid molecules in an emulsion droplet, wherein the artisans teach that the amplification products can be sequenced:
“After encapsulation, the template nucleic acid may be amplified by any suitable method of DNA amplification … DNA amplification is performed by PCR … by exposing the emulsion to any suitable thermocycling regimen known in the art … Following amplification of the template, the emulsion is ‘broken’ (also referred to as ‘demulsification’ in the art).  There are many methods of breaking an emulsion … one preferred method of breaking the emulsion is to add additional oil to cause the emulsion to separate into two phases.  The oil phase is then removed, and a suitable organic solvent (e.g., hexane) is added.  After mixing, the oil/organic solvent phase is removed … Finally, the aqueous layers above the beads are removed.  The beads are then washed with an organic solvent/annealing buffer mixture … Suitable organic solvents include alcohols such as methanol, ethanol, and the like” (section [0192]).
“integrated system, comprising … nucleic acid sample preparation, … nucleic acid amplification, and … DNA sequencing” (section [0016])
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., Benett et al., and Leamon 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 addition, Cho et al. explicitly state that nucleic acid amplification typically precedes analysis such as nucleic acid sequence analysis:
“To disclose the genetic information of nucleic acids such as DNAs and RNAs for the purpose of sequence analysis and disease diagnosis, amplification of trace amounts of nucleic acids to a desired level is required.” (section [0002])
Therefore, one of ordinary skill in the art would have recognized as well as would have been motivated to employ the droplets containing the amplification products of Cho et al. in a nucleic acid sequencing analysis.  Indeed, this motivation in the art is evidenced by Leamon et al. who explicitly teach that nucleic acid templates amplified in droplets should be sequenced (see above).
Leamon et al. also teach the common conclusion that in order to sequence the amplified nucleic acids in the droplets, droplets must first be “broken”.  Leamon et al. teach that this process can be performed by transferring the plurality of droplets and immersing them into a droplet-destabilizing second fluid (additional oil is added as well as a suitable organic solvent), allowing for the amplification products to be liberated and purified from the droplets and subsequently sequenced.
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 1 574 5863 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003) 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).
The teachings of Cho et al., Kumacheva et al., Bhagat et al., and Leamon et al. have been discussed above.
Cho et al., Kumacheva et al., Bhagat et al., and Leamon 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.
Claims 41-43  are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 574 5864 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003) as applied to claims 1, 4, 12, 13, 21, 31, 39, and 40 above, and further in view of Viovy et al. (WO 2006/027757 A2, published March 2006, priority September 2004).
The teachings of Cho et al., Kumacheva et al., Bhagat et al., and Leamon et al. have been discussed above.
While Leamon et al. teach that the droplets should be broken for sequencing the amplified products therein, the artisans do not explicitly teach all types of means which could be used for the purpose, and consequently do not teach that oil comprising surfactant (claim 41) is used, wherein said surfactant promote coalescence of the droplets (claim 42), wherein the surfactant comprises a perfluorinated alcohol (claim 43).
Viovy et al. teach that when adding various wt% of a fluorosurfactant, perfluorodecam-1-ol, (Fluorochem), the surfactant reduced the interfactial tension between the droplets, resulting in the destabilization of the droplets and their break (page 35, lines 11-14).
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 Viovy et al., thereby arriving at the invention as claimed for the following reasons.
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.”
The use of surfactants in disrupting emulsion particles had been well-known in the art, as well as evidenced by Viovy et al.  Given that Leamon et al. explicitly suggested that, “one of ordinary skill in the art be able to select the proper method of ... breaking the emulsion” (Leamon et al., section [0192]), employing art known means to disrupt the emulsion particles, such as those disclosed by Viovy et al., would have been obvious to said ordinary skill in the art.
Conclusion
No claims are allowed.
Applicant’s arguments with respect to the previous rejections of record have been considered but are moot in view of the present rejections based on additional references.
Inquiries

APPLICANT REBUTTAL: Remarks
With entry of this amendment, claims 1, 4, 12, 13, 21, 31-34, and 39-43 are pending, with claim 1 being the only independent claim. Claims 1, 40, and 41 are amended. Support for the amendments may be found in the original application at least at: page 69, line 23-page 70, line 12; page 54, lines 4-5; page 86, lines 7-10 ; page 86, line 8; and page 29, lines 5-23.
No new matter is added.
Claim Objections
The Office Action, at page 2, states that claim 41 is objected to where the hyphen should be removed from the word "de-stabilizing".
Claim 41 is amended as suggested. Applicant submits that the objection is overcome.
Rej
ections under 35 U.S.C.
Q
The Office Action, at page 3, states that claim 40 is rejected under § 112 (pre-AIA),
second paragraph, as being indefinite where, in the phrase, "the one droplet of the plurality of droplets" it is unclear which of the many (plurality) of droplets, "the one droplet" is referring to.
Claim 40 is amended to recite "wherein the PCR reaction is performed by sending the plurality droplets through a serpentine path between the heating and cooling lines in the chip." Applicant submits that the rejections under § 112,  2 are overcome.
Rej
ections under 35 U.S.C.
Q
The Office Action, at page 3, states that claims 1, 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, Bhagat, 2007, Re-usable quick-release interconnect for characterization of microfluidic systems, J Micromech Microeng 17:42-3, and US 2006/0040297 to Leamon. At pages 10-11, claims 32- 34 are so rejected over Cho, Kumacheva, Bhagat, Leamon and further in view of Dorfman, 2005, Anal Chem 77:3700-3704. At page 14, claims 41-43 are rejected over Cho, Kumacheva, Bhagat, Leamon, and WO 2006/027757 to Viovy.
Applicant submits that the claims are not obvious over Cho, Kumacheva, Bhagat, Leamon, Dorfman, and Viovy.
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 that includes copying the nucleic acid within an aqueous droplet and then transferring droplets from one carrier fluid into a second, droplet- destabilizing fluid to recover aqueous sample fluid including an amplification product from droplets.
Recovery of the aqueous sample fluid including the amplification product by such means cannot be found among Cho, Kumacheva, Bhagat, Dorfman, and Viovy-however combined.
Kumacheva and Bhagat do not even mention droplets, much less aqueous droplets, and yield nothing probative under the basic factual inquiries required by Graham v. John Deere.
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, [001S]. In Cho, amplification occurs in a reaction channel with a serpentine shape passing between two temperature areas. Id., [0045]. Cho never mentions recovering droplet contents or any version of releasing products from droplets, much less destabilization to recover aqueous droplet contents including PCR products as recited in amended claim 1.
Leamon reports amplifying individual DNA samples in microcapsules of an emulsion and releasing the amplified DNA from the microcapsules for subsequent reactions. US 2006/0040297, [0017]. In Leamon, single templates may be hybridized to DNA capture beads and emulsified into microreactors. Id. Leamon states that, "After PCR, the emulsion is broken to release the beads, which include beads with amplified, immobilized DNA template, and empty beads". Id., [0810]. Specifically, Leamon states that:
Following amplification of the template, the emulsion is "broken" (also referred
to as "demulsification" in the art). There are many methods of breaking an emulsion (see,
e.g., U.S. Pat. No. 5,989,892 and references cited therein) and one of skill in the art
would be able to select the proper method. In the present invention, one preferred method
of breaking the emulsion is to add additional oil to cause the emulsion to separate into
two phases. The oil phase is then removed, and a suitable organic solvent (e.g., hexanes)
is added. After mixing, the oil/organic solvent phase is removed. This step may be
repeated several times. Finally, the aqueous layers above the beads are removed. The
beads are then washed with an organic solvent/annealing buffer mixture (e.g., one suitable annealing buffer is described in the examples), and then washed again in
annealing buffer. Suitable organic solvents include alcohols such as methanol, ethanol
and the like. Id., [0192].
That passage of Leamon does not describe transferring droplets from one carrier fluid into a second, droplet-destabilizing fluid. In fact, that quoted passage from Leamon specifically describes removing oil and then removing the aqueous phase from beads, and subsequently washing the beads with an organic solvent. The aqueous phase is not in any meaningful sense "recovered", and any amplification product remains with the beads, which is the entire point of removing the oil & aqueous phase and washing the beads.
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 states that "each droplet is collected at the outlet and its full 2-pL volume is analyzed by gel electrophoresis". Id., 3702. Leamon and Dorfman, like Cho, do not teach or suggest transferring aqueous droplets including an amplification product into a droplet-destabilizing second fluid to recover aqueous sample fluid including the amplification product.
Viovy does not cure the deficiencies of Cho, Leamon, and Dorfman.
Viovy only reports a device and method for contamination-free droplet manipulation, in particular coalescence, in microchannels or closed microfluidic systems. WO 2006/027757, page 5, lines 13-17. Viovy reports that, when transporting a water droplet in carrier fluid of FC40 plus 1H,1H,2H,2H perfluorodecan-1-ol in a microchannel (where interfacial tension between carrier fluid and water is lower than between water and microchannel), one observes imperfect behavior of droplets and contamination between droplets. Id., page 19, lines 3-5 and 12-17. Viovy states that using silicone treated with fluorosilane increases the difference between water/carrier fluid and water/channel interfacial channel and that no contamination is observed. Id., page 19, lines 18-22. Viovy also states that adding 0.5 to 3% 1H,1H,2H,2H perfluorodecan-1-ol to the carrier fluid suppresses both irregular droplet motion and contamination. Id., page 19, lines 17-31. Viovy separately states that when 1H,1H,2H,2H perfluorodecan-1-ol is added to droplets of tris- borate-EDTA (TBE), the interfacial tension between the droplets and FC-40 is reduced, with the result that the TBE droplets are destabilized and break into many small droplets. Id., page 35,
lines 9-14. In none of those examples, does Viovy describe transferring a set of droplets from one carrier fluid to another. Viovy only compares one experiment run to another. Viovy does not anywhere teach or suggest transferring droplets from one carrier fluid into a second, droplet- destabilizing fluid. Viovy also does not teach or suggest any method or technique to recover aqueous sample fluid including amplification product from droplets, much less by transferring droplets that contain the amplification product from one carrier fluid into a second droplet- destabilizing fluid.
None of the references that have been cited teach or suggest transferring aqueous droplets that include an amplification product from one carrier fluid into a second, droplet-destabilizing fluid to recover aqueous sample fluid including the amplification product. That element of Independent claim 1 cannot be found among the prior art. Because that element of claim 1 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 August 23, 2024.
Preliminary Remark
Claims 2, 3, 5-11, 14-20, 22-30, and 35-38 are canceled.
Information Disclosure Statement
The IDS received on July 25, 2024 is proper and is being considered by the Examiner.
The IDS was received with the fee under 37 CFR 1.17(p).
Claim Objections
The objection made to claim 41 for the minor informality noted in the Office Action mailed on May 23, 2024 is withdrawn in view of the Amendment received on August 23, 2024.
Claim Rejections - 35 USC § 112
The rejection of claim 40 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 May 23, 2024 is withdrawn in view of the Amendment received on August 23, 2024.
Claim Rejections - 35 USC § 103
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 1 574 5861 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003), made in the Office Action mailed on May 23, 2024 is withdrawn in view of the Amendment received on August 23, 2024.
The rejection of claims 32-34 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 574 5862 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003) 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 May 23, 2024 is withdrawn in view of the Amendment received on August 23, 2024.
The rejection of claims 41-43 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Cho et al. (EP 1 574 5863 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), Bhagat et al. (Journal of Micromechanics and Microengineering, 2007, vol. 17, pages 42-49), and Leamon et al. (US 2006/0040297 A1, published February 23, 2006, priority August 2003) as applied to claims 1, 4, 12, 13, 21, 31, 39, and 40 above, and further in view of Viovy et al. (WO 2006/027757 A2, published March 2006, priority September 2004), made in the Office Action mailed on May 23, 2024 is withdrawn in view of the Amendment received on August 23, 2024.
Rejection – New Grounds, Necessitated by IDS
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, 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 1 574 5864 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), and Dressman et al. (WO 2005/010145 A2, published February 2005; IDS ref), priority July 2003).
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), and
copying the nucleic acid by extending the at least one primer (PCR copies the nucleic acid template).
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 has complete isolation from any continuous aqueous phase (claim 4).
Cho et al. do not explicitly state that the emulsion droplets containing the amplification products are transferred from the oil carrier fluid into a droplet-destabilizing second fluid, and the aqueous sample fluid is removed from the plurality of droplets.
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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)
Dressman et al. teach a method of amplifying nucleic acid molecules in an emulsion droplet, wherein the artisans teach that the amplification products can be sequenced:
“method for analyzing nucleotide sequence variations … forming microemulsions comprising one or more species of analyte DNA molecules, amplifying analyte DNA molecules in the microemulsions in the presence of reagent beads …” (claim 35).
Dressman et al. also teach a well-known process of breaking the emulsion particles by adding detergents:
“Emulsion can be ‘broken’ or disrupted by any means known in the art.  One particularly simple way to break the emulsion is to add more detergent … can be used include, but are not limited to Triton X 100, Laureth 4, Nonidet” (section [0035])
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 Dressman 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 addition, Cho et al. explicitly state that nucleic acid amplification typically precedes analysis such as nucleic acid sequence analysis:
“To disclose the genetic information of nucleic acids such as DNAs and RNAs for the purpose of sequence analysis and disease diagnosis, amplification of trace amounts of nucleic acids to a desired level is required.” (section [0002])
Therefore, one of ordinary skill in the art would have recognized as well as would have been motivated to employ the droplets containing the amplification products of Cho et al. in a nucleic acid sequencing analysis.
Indeed, this motivation in the art is evidenced by Dressman et al. who explicitly teach that nucleic acid templates amplified in droplets should be sequenced (see above).
Dressman et al. also teach the common conclusion that in order to sequence the amplified nucleic acids in the droplets, droplets must first be “broken”.  Dressman et al. teach that this process can be performed by adding additional reagents with the plurality of droplets which results in phase separation of the immiscible reagents.  Whether one takes the medium containing the emulsion products containing the amplificons into a droplet-destabilization reagent or visa versa would have been an obvious order as doing so would have resulted in the same “mixing” of the medium responsible for destabilizing the emulsion droplets, resulting in a phase separation.
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 1 574 5865 A2, published 2005) in view of Kumacheva et al. (WO 2005/103106 A1, published November 2005, priority April 2004), and Dressman et al. (WO 2005/010145 A2, published February 2005; IDS ref), priority July 2003), 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).
The teachings of Cho et al., Kumacheva et al., and Dressman et al. have been discussed above.
Cho et al., Kumacheva et al., and Dressman 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 Dressman 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 Dressman 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.
Claims 41-43 are free of prior art, but are objected to for being dependent on a rejected base claim.
The prior art does not teach or suggest a method involving the mixing of droplets of the from a first carrier fluid (i.e., immiscible to aqueous sample fluid, see claim 1) with a droplet-destabilizing second fluid, wherein said destabilizing second fluid comprises an oil comprising a destabilizing surfactant (claim 41), said surfactant comprising perfluorinated alcohol.
Instant application discloses that the use of oil comprising a destabilizing surfactant results in a gentle manner so as to minimize potential damage to the contents of the droplets:
“present invention proposes methods for recovering aqueous phase components from aqueous emulsions that have been collected on a microfluidic device in a minimum number of steps and in a gentle manner” (section [0279])
“a stable aqueous sample droplet emulsion containing phase components in a continuous phase carrier fluid is allowed to cream to the top of the continuous phase carrier oil … aqueous emulsion rises to the top or separates from the continuous phase fluid being greater than that of the aqueous phase emulsion … The creamed emulsion is then placed onto a second continuous phase carrier fluid which contains a de-stabilizing surfactant … the aqueous emulsion begins to coalesce … can now be removed” (sections [0280] and [0281])
Applicant's submission of an information disclosure statement under 37 CFR 1.97(c) with the fee set forth in 37 CFR 1.17(p) on August 23, 2024 prompted the new ground(s) of rejection presented in this Office action.  Accordingly, THIS ACTION IS MADE FINAL.  See MPEP § 609.04(b).  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 US20060040297A1: Claims:
A method for sequencing a nucleic acid, the method comprising: (a) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 μm and the planar surface has at least 10,000 reaction chambers; (b) performing a pyrophosphate based sequencing reaction simultaneously on all reaction chambers by annealing an effective amount of a sequencing primer to the nucleic acid templates and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′ end of said sequencing primer, a sequencing reaction byproduct; and (c) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in each reaction chamber.
(a) providing a plurality of single-stranded nucleic acid templates disposed within a plurality of cavities on a planar surface, each cavity forming an analyte reaction chamber, wherein the reaction chambers have a center to center spacing of between 20 to 100 Î¼m and the planar surface has at least 10,000 reaction chambers;
(b) performing a pyrophosphate based sequencing reaction simultaneously on all reaction chambers by annealing an effective amount of a sequencing primer to the nucleic acid templates and extending the sequencing primer with a polymerase and a predetermined nucleotide triphosphate to yield a sequencing product and, if the predetermined nucleotide triphosphate is incorporated onto the 3′ end of said sequencing primer, a sequencing reaction byproduct; and
(c) identifying the sequencing reaction byproduct, thereby determining the sequence of the nucleic acid in each reaction chamber.
The method of claim 1 , wherein the sequencing reaction is performed in the presence of apyrase.
The method of claim 2 wherein said apyrase is in solution.
The method of claim 2 wherein said apyrase is immobilized on a surface in said analyte reaction chamber or immobilized on a mobile solid support disposed at each analyte reaction chamber.
The method of claim 1 wherein said pyrophosphate based sequencing reaction is performed at 35° C.
Ref WO2006027757A2: Claims:
A microfluidic device (1) for deforming, in particular splitting, at least one packet, or displacing at least two packets towards each other, in particular for collapsing said packets, said device comprising: - a microchannel (2) having an axis (X),
- packet manipulation means comprising at least one of:
â¢ a generator unit (9), and an electrode assembly (3) coupled to the generator unit (9) and configured for creating inside at least one portion of the microchannel an electric field which is substantially collinear to the axis (X) of the microchannel, wherein the generator unit (9) is capable of generating the electric field with such an amplitude and frequency that the electric field causes the at least one packet to deform, or the at least two packets to displace towards each other in the microchannel, â¢ at least one side channel with a first end in connection with a portion of said microchannel and a second end in connection with a delivery system suitable for delivering a solution able to alter the interfacial tension between said at least two packets or said at least one packet and the environment thereof, said delivery system being configured to deliver said solution into said portion of the microchannel at least during the passage of said packet(s) in said portion of the microchannel.
A device according to the preceding claim, wherein the at least one portion of the microchannel in which the electric field is substantially collinear to the axis (X) of the microchannel has a length comprised between about 1 and about 100 times the thickness of said portion, and preferably between about 1 to about 10 times the thickness of said portion.
A device according to any preceding claim, wherein the electrode assembly (3) is electrically insulated from the inside surface of the microchannel (2).
A device according to any preceding claim, wherein the electrode assembly (3) comprises at least two electrodes (4; 35) axially spaced along the axis of the  microchannel by a distance long enough for the electric field between the electrodes to be substantially collinear to the axis (X) of the microchannel.
A device according to the preceding claim, wherein at least one of said electrodes (4; 35) comprises at least two equipotential portions facing each other across the microchannel (2).
A device according to any one of claims 4 and 5, wherein at least one of said electrodes (4) is monolithic.
A device according to any one of claims 4 to 6, wherein at least one of said electrodes (4) has a cylindrical surface surrounding the microchannel.
A device according to any one of claims 4 and 6, wherein at least one of said electrodes (35) is composite.
A device according to the preceding claim, wherein at least one of said electrodes (35) comprises at least two substantially parallel equipotential plates (36) sandwiching the microchannel (2).
A device according to any one of claims 4 to 9, wherein the electrodes (4;
35) are spaced by a gap (6) having a length that is greater than the thickness of the microchannel, preferably greater than twice the microchannel thickness.
A device according to any preceding claim, wherein the electrode assembly (3; 3') is configured so that in at least one cross section of the microchannel the amplitude of the electric field varies less than a factor 10, better less than 2, and preferably is substantially uniform.
A device according to any preceding claim, wherein the generator unit is capable of generating a continuous field.
A device according to any of claims 1 to 11, wherein the generator unit is capable of generating a variable field.
A device according to the preceding claim, wherein the generator unit is capable of generating an AC field.
A device according to the preceding claim, wherein the generator unit (9) is capable of generating the AC electric field with a frequency ranging from about 0,01 Hz to about 1 GHz, preferably from about 1 Hz to about 10 MHz.
A device according to the preceding claim, for coalescing droplets, wherein the generator unit (9) is capable of generating the AC electric field with a frequency comprised between 100 Hz and 10 kHz.
A device according to claim 15, for aliquoting at least one droplet, wherein the generator unit (9) is capable of generating the AC electric field with a frequency lower than 50 Hz.
A device according to claim 1, wherein said solution contains a surfactant.
A method for displacing at least one packet in a microchannel having an axis, said method comprising: - introducing at least one packet in the microchannel, generating an electric field within at least one portion of the microchannel, at least when the at least one packet is located within said portion, said electric field being preferably collinear to the axis of the microchannel, such as to displace the packet along the microchannel.
Method for collapsing at least two packets in a microchannel (2), the microchannel having a longitudinal axis (X), said method comprising: introducing the at least two packets in the microchannel, generating an electric field within at least one portion of the microchannel, at least when the packets are located within said microchannel portion, said electric field being preferably substantially collinear to the axis of the microchannel in said portion and having an amplitude and a frequency chosen such as to displace two packets towards each other.
Method according to the preceding claim, the electric field being generated by at least two electrodes (4; 35) axially spaced along the axis (X) of the microchannel, said electrodes being separated by a gap (6), the method comprising: before generating said electric field, positioning two packets in the gap between the electrodes, said packets being in static equilibrium, generating said electric field.
Method according to claim 20, the electric field being generated by at least two electrodes (4; 35) axially spaced along the axis of the microchannel, said electrodes being separated by a gap, the method comprising:  positioning two packets in the microchannel, at least one of which being outside the gap between the electrodes, displacing the packets towards the gap, for example via a flowing stream in the microchannel, - generating said electric field at least when the packets are located in the gap-
Method according to any of claims 20 to 22, wherein at least one of said packets contains biological material.
Method for collapsing at least two packets in a microchannel, or for splitting at least one packet, the microchannel having an axis, said method comprising: positioning the at least two packets or the at least one packet in a portion of the microchannel, delivering into said portion of the microchannel a solution of a surfactant able to alter the interfacial tension between said at least two packets or said at least one packet and the environment thereof.
A method for splitting at least one packet in a microchannel having a longitudinal axis, said method comprising: introducing the at least one packet in the microchannel, generating an electric field within at least one portion of the microchannel, at least when the at least one packet is located within said at least one portion, said electric field being preferably substantially collinear to the axis of the microchannel in said portion and having an amplitude and a frequency chosen such as to split the packet.
A method for performing at least one operation on at least one packet in a micro-container, in particular a microchannel, wherein said micro-container has at least one tubular portion defining an internal space of the micro-container, wherein: the tubular portion is made of a non internally coated bulk fluorinated material, or the tubular portion is made of a bulk non- fluorinated material coated on all a circumference of an internal surface of the tubular portion with a permanent layer, wherein said permanent layer is preferably hydrophobic, and  wherein said micro-container is at least partially filled with a carrier fluid immiscible with said packet and containing at least one surfactant at a concentration large enough to decrease the surface tension between said packet and said carrier fluid.
A method according to claim 26, wherein the micro-container comprises a succession of at least two tubular portions, a first tubular portion made of a bulk fluorinated material and a second tubular portion made of a bulk non-fluorinated material coated on all an inner circumference with a permanent layer.
A method according to claim 26 or 27, wherein the permanent layer comprises a material selected among: fused silica glass, PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), any kind of elastomer or plastic, such as for example polyethylene, polyimide, epoxy, Teflon Â® , Parylene Â® , polystyrene, polyethylene terephtalate, polyester, cyclic olefin copolymer, non-conducting oxide such as, for example, glass, silicon dioxide, diamond, non-conductive ceramics, a silicone, an elastomer, a glassy material, a mineral material, a ceramic, a polymer, a thermoplastic polymer, a thermocurable resin, a photocurable resin, a copolymer, a silane, a fluorosilane, a fluoropolymer.
A method according to anyone of claims 26 to 28, wherein the bulk material is selected among: fused silica glass, PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), any kind of elastomer or plastics, such as for example polyethylene, polyimide, epoxy, Teflon Â® , Parylene Â® , polystyrene, polyethylene terephtalate, fluoropolymer, polyester, cyclic olefin copolymer, non-conducting oxide such as, for example, glass, silicon dioxide, diamond, non-conductive ceramics, a silicone, an elastomer, a glassy material, a mineral material, a ceramic, a polymer, a thermoplastic polymer, a thermocurable resin, a photocurable resin, a copolymer.
A method for performing at least one operation on at least one packet in a micro-container, in particular a microchannel, wherein said micro-container has an inner tubular hydrophobic surface, wherein said micro-container is at least partially filled with a carrier fluid immiscible with said packet and containing at least one surfactant at a concentration large enough to decrease the surface tension between said packet and said carrier fluid.
A method according to claim 30, wherein the operation is at least one of displacing the at least one packet, splitting the at least one packet, coalescing the at least one packet with at least another packet, reacting the at least one packet.
A method according to claim 31, wherein the displacing comprises circulating the carrier fluid in the micro-container.
A method according to any one of the claims 30 to 32, wherein the operation is carried out in the absence of any electrical field.
A method according to any one of the claims 30 to 33, wherein the operation comprises displacing the at least one packet from an inlet of the micro-container towards an outlet of the micro-container.
A method according to any one of the claims 30 to 34, wherein the operation comprises exposing successively the at least one packet to at least two different physical and/or chemical conditions, in particular to at least two different temperatures.
A method for performing at least one operation on at least one packet in a micro-container, in particular in a microchannel, said micro-container having an inner surface, wherein said micro-container is filled with a carrier fluid immiscible with said packet and containing at least one surfactant, wherein the difference between the interfacial tension between the packet and the inner wall of the micro-container and the interfacial tension between the packet and the carrier fluid is at least 26 mN/m, preferably at least 35 mN/m, said difference being for instance comprised between about 35 mN/m and about 45mN/m.
A method according to claim 36, wherein the concentration of the surfactant in the carrier fluid is comprised between about 0.01% and about 10% w/w (weight by weight), preferably between about 0.1% and about 3%.
A method according to claim 36 or 37, wherein said surfactant is a fluorosurfactant, in particular a fluoroalcohol.
A method according to claim 38, wherein the fluorosurfactant is 1H,1H,2H,2H Ïerfluorodecan-1-ol.
A method according to any of claims 36 to 39, wherein the bulk portion is silanized.
A device for performing at least one chemical, physical or biological operation on at least one packet embedded in a carrier fluid immiscible with said packet,  said device comprising at least a micro-container surrounding said carrier fluid containing said packet, wherein the inner surface of said micro-container is fluorinated, and said carrier fluid contains a surfactant at a ratio concentration at least 0.1 cmc (critical micellar concentration).
A microfluidic device comprising a micro-container, in particular a microchannel, said device further comprising a tubular bulk hydrophobic portion forming an internal space of the micro-container wherein said bulk portion is coated with a hydrophobic layer.
A connector allowing contamination free transport of at least one packet from at least one microchannel towards another microchannel, said connector comprising, preferably being constituted, by a bulk material having on all its inner surface an hydrophobic layer, said layer comprising one of a fluorinated material and a silanized material.
A connector according to claim 43, wherein said bulk material is an elastomer.
A microfluidic device comprising at least one micro-container, in particular at least one microchannel, and a connector in communication with the micro- container, said connector being configured for connecting said micro-container to at least one of another micro-container, in particular a microchannel, and an inlet or outlet port of the device, wherein said connector has an inner surface comprising at least one hydrophobic layer.
A connector configured for being mounted at one end of a capillary tube forming a microchannel, wherein said connector has an inner surface comprising at least one hydrophobic layer.
A kit comprising: a microfluidic device comprising a microchannel, a connector as disclosed above, to be mounted on said microfluidic device.
A kit for performing at least an operation on a packet comprising: a micro-container, in particular a microchannel, having an inner surface comprising at least one hydrophobic material,  a carrier fluid immiscible with said packet containing at least one surfactant at a concentration large enough to decrease the surface tension between said packet and said water-immiscible fluid.
An assembly comprising: - a connector according to claim 43, at least one capillary tube connected to said connector.
An assembly according to claim 49, wherein the connector is a T- connector.
An assembly according to claim 49 or 50, wherein the capillary tube has a fluorinated inner surface.
A device for performing a PCR comprising: a microchannel comprising a coil made of a capillary tube at least partly filled of a fluorosolvent containing a surfactant, the coil comprising a denaturing region, an annealing region and an elongation region exposed to different temperatures.
A method according to claim 1, for performing at least one of: mixing, nucleic acid screening, nucleic acid amplification, e.g. by PCR, NASBA, rolling circle amplification - RNA reverse transcription
- genotyping, proteomic analysis, transcriptome analysis, crystallization, and in particular protein crystallisation, - searching and evaluation of pharmaceutical targets, pharmaceutical hits or leads, or drugs, enzyme-protein reaction, antigen-antibody reaction, screening of libraries of chemical of biological products, - high throughput screening, drag delivery, diagnosis,  analysis or lysis of at least one living cell or dead cell, analysis of microorganisms, chemical reaction, reactive-catalyzer reaction, - polymerization reaction, fusing particles, for example colloids, to form a chain, preparation of colloids, emulsions, vesicles, in particular monodisperse colloidal objects, preparation of nanoparticules or microparticles, - environmental control, detection of pollutants, - control of an industrial process.
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 micr
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