Office Action 14: Final Rejection (2024-10-21)
Rejection types: 103 - Obviousness

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 first carrier fluid via at least one oil supply line;
applying a pressure to flow the immiscible first carrier fluid into a main channel on the microfluidic substrate;
adjusting pressure in the at least one inlet channel, thereby producing a plurality of droplets comprising the aqueous sample fluid in the main channel;
copying the nucleic acid within one droplet of the plurality of droplets by extending the at least one primer to generate an amplification product; and
transferring the plurality of droplets from the first carrier fluid into a droplet-destabilizing second fluid, thereby recovering the aqueous sample fluid including the amplification product from the plurality of droplets.

2-3. (Cancelled)

4\. (Previously Presented) 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\. (Previously Presented) The method of claim 1, wherein the aqueous sample fluid further comprises 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.
14-20. (Cancelled)1519

21\. (Currently Amended) The method of claim 1, wherein the at least one oil supply line provides a constant flow of the first carrier fluid into a \[\[the\]\] nozzle while the plurality of droplets are formed.

22-30. (Cancelled)2329

31\. (Currently Amended) The method of claim 1, wherein the first 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.

40\. (Currently Amended) The method of claim 39, wherein the PCR reaction is performed by sending the plurality droplets through a serpentine path between the heating and cooling lines in the chip.

41\. (Currently Amended) The method of claim 1, wherein the droplet-destabilizing second fluid is an oil comprising a destabilizing surfactant.
42\. (Previously Presented) 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\. (Currently Amended The method of claim 41, wherein the destabilizing surfactant comprises a perfluorinated alcohol.

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

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
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 16, 2025 has been entered.
Preliminary Remark
Claims 2, 3, 5-11, 14-20, 22-30, 35-38, and 41 are canceled.
Claims 44-50 are new.
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 Dressman et al. (WO 2005/010145 A2, published February 2005; IDS ref), priority July 2003), made in the Office Action mailed on October 21, 2024 is withdrawn in view of the Amendment received on January 16, 2025.
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), 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), made in the Office Action mailed on October 21, 2024 is withdrawn in view of the Amendment received on January 16, 2025.
Conclusion
Claims are free of prior art.
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, said surfactant comprising perfluorinated alcohol.
Instant application discloses that the use of oil comprising a destabilizing surfactant result 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])
Inquiries

CITED REFERENCE SUMMARIES: Ref US6428053B1: Claims:
A method of fabricating a micromachined coupler for coupling a capillary having a first size to an orifice having a shape and a second size comprising:
masking a substrate from which a body of said micromachined coupler will be formed, said masking forming a pattern to define a pyramidal structure in said substrate;
anisotropically etching said masked substrate to form said pyramidal structure including a pit in said pyramidal structure, where anisotropically etching said masked substrate to form said pyramidal structure forms a truncated structure with a basal shoulder;
masking a surface of substrate opposing said pyramidal structure; and
defining a prismatic hole through said substrate and communicating with said pit to provide a through hole through said substrate.
The method of claim 1 where anisotropically etching said masked substrate to form said pyramidal structure forms a simple truncated structure.
The method of claim 1 further comprising disposing a capillary into said prismatic hole in a sealed relationship therewith.
The method of claim 1 where anisotropically etching said masked substrate to form said pyramidal structure forms a square truncated structure.
The method of claim 1 where anisotropically etching said masked substrate to form said pyramidal structure forms a triangular truncated structure.
The method of claim 1 where anisotropically etching said masked substrate to form said pyramidal structure forms a conical truncated structure.
anisotropically etching said masked substrate to form said pyramidal structure including a pit in said pyramidal structure;
defining a prismatic hole through said substrate and communicating with said pit to provide a through hole through said substrate, where defining a prismatic hole through said substrate does not completely remove said pit so that a tubing stop is formed by a remaining portion of said pit.
defining a truncated pyramidal cavity in a micromachined mold;
disposing a material is said truncated pyramidal cavity to form a body of said coupler; defining by a prismatic hole of a first size through said body to define a through hole by deep reactive ion etching; and
defining by deep reactive ion etching a prismatic hole of a second size in said body aligned with said prismatic hole of said first size to define a tubing stop in said prismatic hole of said first size.
Ref US6832787B1: Claims:
A fluid connector for coupling at least one fluid conduit to a corresponding port of a substrate having a network of microchannels, said fluid connector comprising:
a manifold comprising one or more channels extending therethrough wherein each channel is at least partially threaded and each channel has a tapered configuration;
one or more externally threaded ferrules each defining a bore extending therethrough with each ferrule supporting a fluid conduit wherein each ferrule is threaded into a channel of the manifold and wherein each ferrule defines a lower tapered exterior end;
a substrate having one or more ports on its upper surface wherein the substrate is positioned below the manifold so that the one or more ports is aligned with the one or more channels of the manifold; and
means for applying an axial compressive force to the substrate to couple the one or more ports of the substrate to a corresponding proximal end of a fluid conduit; and
a base plate that is rigidly fastened to and spaced apart from manifold and which is positioned below the substrate; and a pressure plate that is interposed between the substrate and the base plate.
The fluid connector of claim 1 wherein the pressure plate includes one or more adjustable screws for maintaining an upward axial force on the substrate.
The fluid connector of claim 2 wherein the manifold and pressure plate define a slot into which the substrate is positioned and the fluid connector comprises means for positioning the pressure plate.
The fluid connector of claim 3 wherein the means for positioning the pressure plate comprises at least one guide pin which is disposed on the base plate.
The fluid connector of claim 1 wherein the substrate is a microfluidic device.
The fluid connector of claim 1 each fluid conduit comprises a capillary tube having an inner diameter that ranges from 5 to 250 microns.
The fluid connector of claim 1 wherein the lower surface of the manifold defines one or more channel outlets and the lower surface adjacent each outlet defines an annular groove that contains a seal material around the outlet.
The fluid connector of claim 7 wherein the seal material is an o-shaped ring made of a material selected from rubber and silicone.
The fluid connector of claim 1 wherein each ferrule is an integral structure with no mating sleeve.
The fluid connector of claim 1 wherein each ferrule is made of a deformable material.
The fluid connector of claim 1 wherein a portion of each ferrule is sufficiently compressed against a part of a fluid conduit to form a fluid tight seal.
The fluid connector of claim 1 wherein each ferrule comprises an adapter bode having a nut on one side and having an elongated member on the other side and wherein the bore extends through the adapter body, nut and elongated member.
The fluid connector of claim 12 wherein the lower tapered exterior end is on the elongated member.
The fluid connector of claim 12 wherein the part of the bore that extends through the nut has a diameter that is larger than the part of the bore that extends through the elongated member.
The fluid connector of claim 13 wherein each ferrule is an integral structure with no mating sleeve.
The fluid connector of claim 1 wherein each channel defines a conical-shaped cavity.
The fluid connector of claim 1 wherein the means for applying the axial compressive force enables the fluid connector o operate at a pressure of at least about 500 psi.
The fluid connector of claim 1 wherein each ferrule comprises an upper handle, an externally threaded portion, and wherein the bore extends through the upper handle, externally threaded portion, and the lower tapered exterior end.
The fluid connector of claim 18 wherein the part of the bore that extends through the tapered end has a diameter that is narrower than the part of the bore that extends through the handle.
The fluid connector of claim 18 wherein each ferrule is an integral structure with no mating sleeve.
Ref US7604938B2: Claims:
A method of detecting the absence or presence of one or more organisms in one or more samples, comprising the steps of: (a) providing a microfluidic device comprising a microfluidic reagent assembly area which forms two or more sample plugs, said microfluidic reagent assembly area being in fluid communication with at least one microfluidic inline channel that runs through an amplification area and a detection area, said microfluidic reagent assembly area comprising: i. a sample droplet forming area which receives one or more samples containing the same or different genomic material and forms one or more samples into said one or more sample droplets, said sample droplet forming area comprises a sample droplet microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more sample droplets to said microfluidic inline channel; ii. a primer plug producing area comprising a primer assembly apparatus which assembles one or more primer plugs comprising amplification reagents including at least one primer, wherein said primer assembly apparatus is controlled to assemble said one or more primer plugs containing the same primers or different primers, said primer plug producing area further comprises a primer plug microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more primer plugs to the microfluidic inline channel; and iii. a sample plug mixing area which forms sample plugs by mixing said sample droplet and primer plug, said sample plug mixing area being in fluid communication with the microfluidic inline channel; wherein said microfluidic reagent assembly area is controlled to form sample plugs that comprise the same or different genomic material and the same or different primers; (b) acquiring samples that may contain an organism; (c) selecting one or more samples to input into said sample droplet forming area of the device; (d) isolating at least one copy of the genomic material if present from each of the selected samples in the sample droplet forming area; (e) introducing at least one sample droplet of each of the selected samples into the microfluidic inline channel through the sample droplet microfluidic path; (f) driving the sample droplets along the microfluidic inline channel through the reagent assembly area; (g) selecting two or more primer plugs to input into the primer plug microfluidic path of the device; (h) assembling the selected primer plugs in the primer plug assembly area, wherein each primer plug comprises amplification reagents and at least one primer, and wherein the primer plugs contain the same primers or different primers; (i) introducing the primer plugs into the microfluidic inline channel through the primer plug microfluidic path; (j) forming two or more sample plugs in the microfluidic inline channel by mixing the sample droplets with the primer plugs in the microfluidic inline channel in the sample plug mixing area, wherein the sample plugs comprise the same or different genomic material and the same or different primers; (k) driving the sample plugs along the microfluidic inline channel through the amplification area; (l) amplifying the genomic material, if present, in each of the sample plugs in the microfluidic inline channel in the amplification area of the device; (m) driving the sample plugs along the microfluidic inline channel through the detection area; and (n) detecting the absence or presence of amplified products in each of the sample plugs, wherein the presence of amplified products for an organism is indicative of the presence of the organism in the sample.
(a) providing a microfluidic device comprising a microfluidic reagent assembly area which forms two or more sample plugs, said microfluidic reagent assembly area being in fluid communication with at least one microfluidic inline channel that runs through an amplification area and a detection area, said microfluidic reagent assembly area comprising: i. a sample droplet forming area which receives one or more samples containing the same or different genomic material and forms one or more samples into said one or more sample droplets, said sample droplet forming area comprises a sample droplet microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more sample droplets to said microfluidic inline channel; ii. a primer plug producing area comprising a primer assembly apparatus which assembles one or more primer plugs comprising amplification reagents including at least one primer, wherein said primer assembly apparatus is controlled to assemble said one or more primer plugs containing the same primers or different primers, said primer plug producing area further comprises a primer plug microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more primer plugs to the microfluidic inline channel; and iii. a sample plug mixing area which forms sample plugs by mixing said sample droplet and primer plug, said sample plug mixing area being in fluid communication with the microfluidic inline channel;
i. a sample droplet forming area which receives one or more samples containing the same or different genomic material and forms one or more samples into said one or more sample droplets, said sample droplet forming area comprises a sample droplet microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more sample droplets to said microfluidic inline channel;
ii. a primer plug producing area comprising a primer assembly apparatus which assembles one or more primer plugs comprising amplification reagents including at least one primer, wherein said primer assembly apparatus is controlled to assemble said one or more primer plugs containing the same primers or different primers, said primer plug producing area further comprises a primer plug microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more primer plugs to the microfluidic inline channel; and
iii. a sample plug mixing area which forms sample plugs by mixing said sample droplet and primer plug, said sample plug mixing area being in fluid communication with the microfluidic inline channel;
wherein said microfluidic reagent assembly area is controlled to form sample plugs that comprise the same or different genomic material and the same or different primers;
(b) acquiring samples that may contain an organism;
(c) selecting one or more samples to input into said sample droplet forming area of the device;
(d) isolating at least one copy of the genomic material if present from each of the selected samples in the sample droplet forming area;
(e) introducing at least one sample droplet of each of the selected samples into the microfluidic inline channel through the sample droplet microfluidic path;
(f) driving the sample droplets along the microfluidic inline channel through the reagent assembly area;
(g) selecting two or more primer plugs to input into the primer plug microfluidic path of the device;
(h) assembling the selected primer plugs in the primer plug assembly area, wherein each primer plug comprises amplification reagents and at least one primer, and wherein the primer plugs contain the same primers or different primers;
(i) introducing the primer plugs into the microfluidic inline channel through the primer plug microfluidic path;
(j) forming two or more sample plugs in the microfluidic inline channel by mixing the sample droplets with the primer plugs in the microfluidic inline channel in the sample plug mixing area, wherein the sample plugs comprise the same or different genomic material and the same or different primers;
(k) driving the sample plugs along the microfluidic inline channel through the amplification area;
(l) amplifying the genomic material, if present, in each of the sample plugs in the microfluidic inline channel in the amplification area of the device;
(m) driving the sample plugs along the microfluidic inline channel through the detection area; and
(n) detecting the absence or presence of amplified products in each of the sample plugs,
wherein the presence of amplified products for an organism is indicative of the presence of the organism in the sample.
The method of claim 1 , wherein the at least one primer comprises a PCR primer pair.
The method of claim 1 , wherein the at least one primer comprises a waveform profiling primer or a single genome profiling (SGP) primer.
The method of claim 1 , wherein the detecting step of step (n) occurs at one temperature.
The method of claim 1 , wherein the detecting step of step (n) occurs at a range of temperatures.
The method of claim 1 , wherein the amplification reagents comprise nucleotides, DNA polymerase, magnesium and a buffer.
The method of claim 6 , wherein the amplification reagents further comprise a detection agent.
The method of claim 1 , wherein multiple sample droplets from one or more samples are introduced into the microfluidic inline channel of the device.
The method of claim 8 , wherein the multiple sample droplets are from one sample.
The method of claim 8 , wherein primer plugs containing different primers are mixed with the multiple sample droplets to form multiple sample plugs in the microfluidic inline reaction channel of the device.
The method of claim 10 , wherein the different primers are capable of amplifying genomic material of different organisms.
The method of claim 8 , wherein the multiple sample droplets are from two or more samples.
The method of claim 12 , wherein primer plugs containing the same primers are mixed with the multiple sample droplets to form multiple sample plugs in the microfluidic inline reaction channel of the device.
The method of claim 12 , wherein primer plugs containing different primers are mixed with the multiple sample droplets to form multiple sample plugs in the microfluidic inline reaction channel of the device.
The method of claim 14 , wherein the different primers are capable of amplifying genomic material of different organisms.
The method of claim 1 , wherein the one or more samples are from a sample supply that is continuously selected and wherein the presence of an organism in the sample indicates contamination of the sample supply.
The method of claim 1 , which further comprises identifying the one or more organisms on the basis of the amplified products and the primers used to amplify the sample plugs.
The method of claim 1 , wherein the sample droplet forming area further comprises a genomic isolation area comprising a filtering apparatus, a genomic material isolation apparatus and a genomic material concentration adjusting apparatus.
A method of detecting the absence or presence of an organism in a sample comprising the steps of: (a) providing a microfluidic device comprising a microfluidic reagent assembly area which forms two or more sample plugs, said microfluidic reagent assembly area being in fluid communication with at least one microfluidic inline channel that runs through an amplification area and a detection area, said microfluidic reagent assembly area comprising: i. a sample droplet forming area which receives one or more samples containing the same or different genomic material and forms one or more samples into said one or more sample droplets, said sample droplet forming area comprises a sample droplet microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more sample droplets to said microfluidic inline channel; ii. a primer plug producing area comprising a primer assembly apparatus which assembles one or more primer plugs comprising amplification reagents including at least one primer, wherein said primer assembly apparatus is controlled to assemble said one or more primer plugs containing the same primers or different primers, said primer plug producing area further comprises a primer plug microfluidic path in fluid communication with said microfluidic inline channel which forwards the one or more primer plugs to the microfluidic inline channel; and iii. a sample plug mixing area which forms sample plugs by mixing said sample droplet and primer plug, said sample plug mixing area being in fluid communication with the microfluidic inline channel; wherein said microfluidic reagent assembly area is controlled to form sample plugs that comprise the same or different genomic material and the same or different primers; and wherein the microfluidic device further comprising at least one microfluidic inline channel that runs through (i) an amplification area within a first temperature-controlled area and (ii) a detection area within a second temperature-controlled area, wherein the at least one microfluidic inline channel comprises a valve downstream of the detection area through which a sample selectively flows and wherein the device controls fluid movement within the microfluidic inline channel, heating and cooling of the first and second temperature-controlled areas and data acquisition from the microfluidic device; (b) acquiring a sample that may contain an organism; (c) isolating at least one copy of the genomic material of the organism, if present in the sample, in the reagent assembly area of the micro fluidic device; (d) introducing at least one sample droplet into one microfluidic inline reaction channel; (e) driving the at least one sample droplet along the microfluidic inline channel through the r
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