Office Action 1: Non-Final Rejection (2018-12-14)
Rejection types: 102 - Lack of Novelty, 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
CLM 15480739 2 2017-04-06 Attorney Docket No.: RDT-514/US07 29168/328 Claims What is claimed is:
1\. A method for droplet formation, the method comprising the steps of: providing a plurality of aqueous fluids in a plurality of sample inlet channels. providing an immiscible carrier fluid in a main channel, wherein each of the sample inlet channels is in fluid communication with the main channel ; forming a plurality of droplets comprising at least one aqueous fluid surrounded by the immiscible carrier fluid in the main channel; applying one pressure to flow the immiscible carrier fluid in the main channel; and adjusting pressure in one or more of the sample inlet channels, thereby to produce a plurality of droplets comprising at least one aqueous fluid in the main channel. Independent
2\. The method of claim 1, wherein the pressure in the main channel is derived from pressure in a reservoir in fluid communication with the main channel.Dependent
3\. The method of claim 1, wherein the pressure in the main channel is applied by a single pressure source to the main channel.Dependent
4\. The method of claim 3, wherein the pressure source comprises a carrier oil.Dependent
5\. The method of claim 1, in the forming step, wherein a plurality of droplets comprising a first aqueous fluid and a plurality of droplets comprising a second fluid are formed.Dependent
6\. The method of claim 5. wherein the droplets comprising a first aqueous fluid and the droplets comprising a second aqueous fluid are substantially uniform in size.Dependent
7\. The method of claim 5, wherein the droplets comprising a first aqueous fluid are of a different size than the droplets comprising a second aqueous fluid.Dependent -113- Attorney Docket No.: RDT-514/US07 29168/328
8\. The method of claim 6, further comprising detecting the size of the droplets.Dependent
9\. The method of claim 1, wherein the immiscible carrier fluid is an oil.Dependent
10\. The method of claim 9, wherein the oil comprises a surfactant.Dependent
11\. The method of claim 10, wherein the surfactant is a fluorosurfactant.Dependent
12\. The method of claim 1, wherein the aqueous fluid comprises nucleic acid and reagents for conducting an amplification reaction.Dependent
13\. The method of claim 12, wherein the amplification reaction is a PCR reaction.Dependent
14\. The method of claim 1, wherein each droplet comprises one or more molecules.Dependent
15\. The method of claim 14, wherein the one or more molecules are selected from the group consisting of a tissues, a cell, a particle, a protein, an antibody, an amino acids, a nucleotide, and any combination thereof. Dependent
16\. The method of claim 1, wherein the droplets are of different sizes.Dependent
17\. The method of claim 1, wherein the droplets are of a highly uniform size.Dependent
18\. The method of claim 9, wherein the oil is a fluorocarbon oil. -114- Dependent

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.
Information Disclosure Statement
No IDS has been filed to date of the instant Office communication.
Priority
The effective priority date established for the present application is determined to be the actual filing date of the instant application, April 6, 2017 because the parent application does not have a proper support for a method involving a device having a plurality of sample inlet channels connected to a main channel carrying an immiscible carrier fluid.
Applicants are requested to point to the line and page numbers of the parent applications to which priority claim is sought for support.
Drawings
The drawings received on June 19, 2017 are acceptable.
Claim Rejections - 35 USC § 112
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 8 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 pre-AIA  the applicant regards as the invention.
Claim 8 recites the phrase, “the droplets”.
It is unclear whether the droplets are referring to the droplets of the first fluid or those of the second fluid.  For the purpose of prosecution, it is assumed that the droplets of the both fluid are being referenced.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of pre-AIA  35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
Claims 1-5, 9, 10, and 12-15 are rejected under pre-AIA  35 U.S.C. 102(b) as being anticipated by Froehlich et al. (US 2014/0065631 A1, published March 2014).
Froehlich et al. disclose a system comprising the below structure (reproduced from Figure 5):
PNG    media_image1.png    390    697    media_image1.png    GreyscaleAs it can be seen, the device comprises a main channel (white arrow), which is disclosed as carrying an immiscible oil (“first reservoir (101) for uptake of a hydrophobic liquid … comprises at least one inlet (102) … with a first microfluidic channel (103)”, section [0011]), this inlet channel is equivalent to the main channel with white arrow indication of the flow direction.  The main channel carrying the hydrophobic liquid is then connected to a plurality of sample inlet channels (indicated black) injecting the samples therein into the main channel carrying the hydrophobic liquid, resulting in the generation of emulsion or microdroplet particles (“at least one element (110) comprising a second reservoir (111) for uptake of a hydrophilic solution ending in a vertical inlet (112) being opening into a second microfluidic channel (113) … connection between the first (103) and the second  microfluidic channel (113) is designed as a T-junction”, section [0011]).  This structure in the embodiment disclosed in Figure 5 (seen above) is the main channel with hydrophobic liquid which connects to a plurality of T-junctions form between each of the sample inlet channels with hydrophilic solution.
Therefore, Froehlich et al. disclose a method of providing a plurality of aqueous fluids in a plurality of inlet channels, providing an immiscible carrier fluid in a main channel, wherein each of the sample inlet channels is in fluid communication  with the main channel, and flowing a plurality of droplets comprising at least one aqueous fluid surrounded by the immiscible carrier fluid in the main channel.
Froehlich et al. explicitly disclose that the flow rate of the first channel is adjusted to control the droplet sizes (“flow rate in the first and second microfluidic channel is adjusted such that the diameter of each aqueous droplet generated within said first microfluidic channel ranges from 10 to 100 m”, section [0063]).
With regard to claims 2-4, Froehlich et al. also disclose that the pressure is applied to follow the immiscible fluid in the main channel (“means for providing a flow within said first microfluidic channel…”, section [0044]; “said first means is a pressure pump…”, section [0045]).
With regard to claim 5, the plurality of sample inlets result in generation of plurality of droplets comprising a first aqueous solution and a plurality of droplets comprising a second aqueous fluid (see Figure 5 above).
With regard to claim 9, the immiscible carrier fluid is oil (“said hydrophobic liquid comprise any oil”, section [0041]).
With regard to claim 10, the oil comprises a surfactant (“emulsion oils and optionally a surfactant”, section [0083]).
With regard to claims 12 and 13, the method involves nucleic acid amplification which is PCR (“present description is further directed to a system for digital PCR”, section [0048]).
With regard to claim 14, each droplet comprises one or more molecules (“said hydrophilic solution … mixture comprising a nucleic acid sample”, section [0040]).
With regard to claim 15, the one or more molecules are nucleotide (“hydrophilic solution comprises … dNTPs”, section [0040]).
Therefore, Froehlich et al. anticipate the invention as claimed.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 6-8, 16, and 17 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Froehlich et al. (US 2014/0065631 A1, published March 2014) in view of Cho et al. (US 2005/0202489 A1, published September 15, 2005).
The teachings of Froehlich et al. have already been discussed above.
While Froehlich et al. explicitly teach that the flow rate of the pump can be adjusted to control the droplet sizes, the artisans do not explicitly disclose that the droplets generated from the multiple inlet channels are of the same size or different size.
However, Cho et al. disclose a common knowledge in the art of emulsion PCR that the sizes of the droplets should be uniform when performing PCR (“the aqueous solution droplets have different sizes, which may cause a reproducibility problrm”, section [0015]; “since the reactant aqueous solution passes through the first inlet channel 141 with a constant sectional area at a constant flow rate, the aqueous solution droplets 132 created in the reaction channel 130 have a uniform size”, section [0081]).
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 Froehlich et al. with the teachings of Cho et al., thereby arriving at the invention as claimed for the following reasons.
Initially, Cho et al. clearly evidence that when performing emulsion PCR, the emulsion particles should be of uniform sizes.  Therefore, given the fact that the method and device disclosed by Froehlich et al. are explicitly taught as being capable adjusting for the sizes of the droplets formed in the main channel carrying the immiscible oil, said one of ordinary skill in the art would have been motivated to keep the droplets generated from the single sample inlet to be kept uniform.
In addition, it is a common knowledge in the art that the concentration of the nucleic acids present in the droplets are also controlled by the size of the droplets.
“the concentration of nucleic acids within said sample is adjusted to provide at average one nucleic acid molecule per droplet … said sample is adjusted to one sequence copy in 1 to 100 pL …” (section [0064], Froehlich et al.)
“the flow rate in the first … microfluidic channel is adjusted such that the volume of each aqueous droplet generated within said first microfluidic channel ranges from 5 to 500 pL” (section [0062], Froehlich et al.)
Therefore, one of ordinary skill in the art would have been motivated to adjust the size of the droplets formed from each of the different sample inlets so as to control the amount of starting nucleic acid present in the droplets to optimize the amplification reaction for droplets generated from each of the different samples from their corresponding sample inlets, resulting in either the uniform sizes or different sizes (when comparing droplets generated from different sample inlets).
With regard to claim 8, the read-out instrument is a flow cytometer (“the read-out instrument is a flow cytometry instrument”, section [0050]).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Claims 11 and 18 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Froehlich et al. (US 2014/0065631 A1, published March 2014) in view of Kiss et al. (Analytical Chemistry, 2008, vol. 80, pages 8975-8981).
The teachings of Froehlich et al. have already been discussed above.
While Froehlich et al. disclose that immiscible oil is employed in generating emulsion droplets therein, the artisans do not explicitly disclose all prior art know variations of such immiscible fluids.
Kiss et al. disclose a well-known practice of employing fluorinate oil, such as fluorocarbon oil as the immiscible oil in generating emulsion droplets therein (“we describe a continuous-flow-based real-time PCR system that generates discrete, picoliter-scale PCR reations in a continuous stream of inert fluorinate oil”, page 8976, 1st column).
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 Froehlich et al. with the teachings of Kiss 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.”
Given the fact that fluorinate oil has been known to be used as an immiscible fluid in an emulsion amplification reaction, said one of ordinary skill in the art would have had a clear expectation of success that combining other well-known immiscible carrier fluid in the method disclosed by Froehlich et al. would have yielded the same predictable result of producing a successful amplification reaction.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries


NEXT OFFICE ACTION: DETAILED ACTION
The present Office Action is responsive to the Amendment received on March 13, 2019.
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Priority
The effective priority date established for the present application is determined to be the actual filing date of the instant application, April 6, 2017 because the parent application does not have a proper support for a method involving a device having a plurality of sample inlet channels connected to a main channel carrying an immiscible carrier fluid.
Applicants are requested to point to the line and page numbers of the parent applications to which priority claim is sought for support.
Response to Applicants’ arguments
Applicants contend that proper support for the subject-limitation can be found in the parent application 11/803,101 (herein, “the ‘101 application”) (page 4, Response).
Applicants reference page 19 of the specification of the ‘101 application discloses a main channel carrying an immiscible carrier fluid (page 4, Response) and that page 31 of the ‘101 application discloses a substrate device having a plurality of  sample inlet channels connected to the main channel, referencing lines 1-9 and 9-12 therein.
Page 31 of the ‘101 application is reproduced in its entirety below.
PNG    media_image1.png    1433    993    media_image1.png    Greyscale
As it can be seen, no such support is found.
Next, Applicants reference Figure 1 of the 60/799,834 in the priority chain of the present application.
Applicants generally refer to the figure stating that, “[t]he configuration of a plurality of sample inlet channels connected to a main chain channel is shown” (page 4, Response).
The above-identified provisional application is a three page document with a single Figure, wherein the figure is described as follows:
PNG    media_image2.png    170    825    media_image2.png    Greyscale
There is no apparent support for a providing a plurality of aqueous fluids in a plurality of sample inlet channels, wherein each of said plurality of sample inlet channels are in fluid communication with the main channel which provides an immiscible carrier fluid.
Therefore, Applicants’ arguments are not found persuasive.
Claim Rejections - 35 USC § 112
The rejection of claim 8 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 December 14, 2018 is withdrawn in view of the Amendment received on March 13, 2019.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of pre-AIA  35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
The rejection of claims 1-5, 9, 10, and 12-15 under pre-AIA  35 U.S.C. 102(b) as being anticipated by Froehlich et al. (US 2014/0065631 A1, published March 2014), made in the Office Action mailed on December 14, 2018 is maintained for the reasons of record.
Applicants arguments pertaining to the priority support has not been found persuasive as already discussed above.
Therefore, the reference of record is prior art and the rejection is maintained for the reasons already of record.The Rejection:
Froehlich et al. disclose a system comprising the below structure (reproduced from Figure 5):
PNG    media_image3.png    390    697    media_image3.png    GreyscaleAs it can be seen, the device comprises a main channel (white arrow), which is disclosed as carrying an immiscible oil (“first reservoir (101) for uptake of a hydrophobic liquid … comprises at least one inlet (102) … with a first microfluidic channel (103)”, section [0011]), this inlet channel is equivalent to the main channel with white arrow indication of the flow direction.  The main channel carrying the hydrophobic liquid is then connected to a plurality of sample inlet channels (indicated black) injecting the samples therein into the main channel carrying the hydrophobic liquid, resulting in the generation of emulsion or microdroplet particles (“at least one element (110) comprising a second reservoir (111) for uptake of a hydrophilic solution ending in a vertical inlet (112) being opening into a second microfluidic channel (113) … connection between the first (103) and the second  microfluidic channel (113) is designed as a T-junction”, section [0011]).  This structure in the embodiment disclosed in Figure 5 (seen above) is the main channel with hydrophobic liquid which connects to a plurality of T-junctions form between each of the sample inlet channels with hydrophilic solution.
Therefore, Froehlich et al. disclose a method of providing a plurality of aqueous fluids in a plurality of inlet channels, providing an immiscible carrier fluid in a main channel, wherein each of the sample inlet channels is in fluid communication  with the main channel, and flowing a plurality of droplets comprising at least one aqueous fluid surrounded by the immiscible carrier fluid in the main channel.
Froehlich et al. explicitly disclose that the flow rate of the first channel is adjusted to control the droplet sizes (“flow rate in the first and second microfluidic channel is adjusted such that the diameter of each aqueous droplet generated within said first microfluidic channel ranges from 10 to 100 m”, section [0063]).
With regard to claims 2-4, Froehlich et al. also disclose that the pressure is applied to follow the immiscible fluid in the main channel (“means for providing a flow within said first microfluidic channel…”, section [0044]; “said first means is a pressure pump…”, section [0045]).
With regard to claim 5, the plurality of sample inlets result in generation of plurality of droplets comprising a first aqueous solution and a plurality of droplets comprising a second aqueous fluid (see Figure 5 above).
With regard to claim 9, the immiscible carrier fluid is oil (“said hydrophobic liquid comprise any oil”, section [0041]).
With regard to claim 10, the oil comprises a surfactant (“emulsion oils and optionally a surfactant”, section [0083]).
With regard to claims 12 and 13, the method involves nucleic acid amplification which is PCR (“present description is further directed to a system for digital PCR”, section [0048]).
With regard to claim 14, each droplet comprises one or more molecules (“said hydrophilic solution … mixture comprising a nucleic acid sample”, section [0040]).
With regard to claim 15, the one or more molecules are nucleotide (“hydrophilic solution comprises … dNTPs”, section [0040]).
Therefore, Froehlich et al. anticipate the invention as claimed.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA  35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The rejection of claims 6-8, 16, and 17 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Froehlich et al. (US 2014/0065631 A1, published March 2014) in view of Cho et al. (US 2005/0202489 A1, published September 15, 2005) made in the Office Action mailed on December 14, 2018 is maintained for the reasons of record.
Applicants arguments pertaining to the priority support has not been found persuasive as already discussed above.
Therefore, the reference of record is prior art and the rejection is maintained for the reasons already of record.The Rejection:
The teachings of Froehlich et al. have already been discussed above.
While Froehlich et al. explicitly teach that the flow rate of the pump can be adjusted to control the droplet sizes, the artisans do not explicitly disclose that the droplets generated from the multiple inlet channels are of the same size or different size.
However, Cho et al. disclose a common knowledge in the art of emulsion PCR that the sizes of the droplets should be uniform when performing PCR (“the aqueous solution droplets have different sizes, which may cause a reproducibility problrm”, section [0015]; “since the reactant aqueous solution passes through the first inlet channel 141 with a constant sectional area at a constant flow rate, the aqueous solution droplets 132 created in the reaction channel 130 have a uniform size”, section [0081]).
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 Froehlich et al. with the teachings of Cho et al., thereby arriving at the invention as claimed for the following reasons.
Initially, Cho et al. clearly evidence that when performing emulsion PCR, the emulsion particles should be of uniform sizes.  Therefore, given the fact that the method and device disclosed by Froehlich et al. are explicitly taught as being capable adjusting for the sizes of the droplets formed in the main channel carrying the  immiscible oil, said one of ordinary skill in the art would have been motivated to keep the droplets generated from the single sample inlet to be kept uniform.
In addition, it is a common knowledge in the art that the concentration of the nucleic acids present in the droplets are also controlled by the size of the droplets.
“the concentration of nucleic acids within said sample is adjusted to provide at average one nucleic acid molecule per droplet … said sample is adjusted to one sequence copy in 1 to 100 pL …” (section [0064], Froehlich et al.)
“the flow rate in the first … microfluidic channel is adjusted such that the volume of each aqueous droplet generated within said first microfluidic channel ranges from 5 to 500 pL” (section [0062], Froehlich et al.)
Therefore, one of ordinary skill in the art would have been motivated to adjust the size of the droplets formed from each of the different sample inlets so as to control the amount of starting nucleic acid present in the droplets to optimize the amplification reaction for droplets generated from each of the different samples from their corresponding sample inlets, resulting in either the uniform sizes or different sizes (when comparing droplets generated from different sample inlets).
With regard to claim 8, the read-out instrument is a flow cytometer (“the read-out instrument is a flow cytometry instrument”, section [0050]).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
The rejection of claims 11 and 18 under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Froehlich et al. (US 2014/0065631 A1, published March 2014) in  view of Kiss et al. (Analytical Chemistry, 2008, vol. 80, pages 8975-8981), made in the Office Action mailed on December 14, 2018 is maintained for the reasons of record.
Applicants arguments pertaining to the priority support has not been found persuasive as already discussed above.
Therefore, the reference of record is prior art and the rejection is maintained for the reasons already of record.The Rejection:
The teachings of Froehlich et al. have already been discussed above.
While Froehlich et al. disclose that immiscible oil is employed in generating emulsion droplets therein, the artisans do not explicitly disclose all prior art know variations of such immiscible fluids.
Kiss et al. disclose a well-known practice of employing fluorinate oil, such as fluorocarbon oil as the immiscible oil in generating emulsion droplets therein (“we describe a continuous-flow-based real-time PCR system that generates discrete, picoliter-scale PCR reations in a continuous stream of inert fluorinate oil”, page 8976, 1st column).
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 Froehlich et al. with the teachings of Kiss et al. thereby arriving at the invention as claimed for the following reasons.
number='10'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.”
Given the fact that fluorinate oil has been known to be used as an immiscible fluid in an emulsion amplification reaction, said one of ordinary skill in the art would have had a clear expectation of success that combining other well-known immiscible carrier fluid in the method disclosed by Froehlich et al. would have yielded the same predictable result of producing a successful amplification reaction.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
number='11'	No claims are allowed.
THIS ACTION IS MADE FINAL.  Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action.  In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action.  In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Inquiries

CITED REFERENCE SUMMARIES: Ref US20140065631A1: Claims:
A microfluidic chip for the generation of aqueous droplets for nucleic acid amplification, said microfluidic chip comprising: a. a first reservoir for uptake of a hydrophobic liquid ending in at least one inlet or at least one inlet coming from an external reservoir of hydrophobic liquid, each inlet ending into a first microfluidic channel connecting the at least one first reservoir with a reaction vessel, b. at least one element comprising a second reservoir for uptake of a hydrophilic solution ending in a vertical inlet into a second microfluidic channel connecting the second reservoir with a first microfluidic channel, c. at least one T-junction between said first and second microfluidic channel, characterized in that the end of said second microfluidic channel is joined radial and horizontal to the first microfluidic channel.
a. a first reservoir for uptake of a hydrophobic liquid ending in at least one inlet or at least one inlet coming from an external reservoir of hydrophobic liquid, each inlet ending into a first microfluidic channel connecting the at least one first reservoir with a reaction vessel,
b. at least one element comprising a second reservoir for uptake of a hydrophilic solution ending in a vertical inlet into a second microfluidic channel connecting the second reservoir with a first microfluidic channel,
c. at least one T-junction between said first and second microfluidic channel, characterized in that the end of said second microfluidic channel is joined radial and horizontal to the first microfluidic channel.
The microfluidic chip of claim 1 , wherein said end of the second channel is joined to the first channel perpendicularly.
The microfluidic chip of claim 1 , wherein said first reservoir for uptake of a hydrophobic liquid ends in at least one vertical inlet or in at least one horizontal inlet.
The microfluidic chip of claim 1 , wherein said hydrophilic solution is an aqueous mixture comprising a nucleic acid sample and wherein said hydrophobic liquid comprises an emulsion oil or a mixture of emulsion oils and optionally a surfactant.
A device for the generation of aqueous droplets for nucleic acid amplification, the device comprising a microfluidic chip according to claim 1 , first means for providing a flow within said first microfluidic channel and second means for providing a flow within said second microfluidic channel.
The device of claim 5 further comprising means for holding at least one reaction vessel for collecting the aqueous droplets, wherein the at least one reaction vessel is selected from the group consisting of a single PCR tube, a PCR strip, for example an 8-well strip, and a 96-well plate.
A system for digital PCR, the system comprising: a. a device according to claim 5 , b. a thermocycler instrument, and c. a read-out instrument.
a. a device according to claim 5 ,
b. a thermocycler instrument, and
c. a read-out instrument.
The system of claim 7 , wherein said device, said thermocycler instrument and said read-out instrument are operatively linked to each other.
The system of claim 7 , wherein said read-out instrument is a flow cytometry instrument or an imaging instrument.
The system of claim 7 , wherein said read-out instrument is capable of reading 1,000 to 100,000 droplets per second.
Use of the microfluidic chip, device or system according to claim 1 for performing digital PCR, wherein digital PCR is performed for the detection of rare mutations in excess background.
A method for the generation of aqueous droplets for nucleic acid amplification, the method comprising the steps of: a. providing a hydrophobic liquid in a first reservoir, b. providing a hydrophilic solution in a second reservoir c. generating a first continuous flow of said hydrophobic liquid from said first reservoir through at least one inlet, each inlet ending into a first microfluidic channel, wherein the first microfluidic channel enters into a reaction vessel, d. generating a second continuous flow of said hydrophilic solution from said second reservoir through a second microfluidic channel, wherein the end of said second microfluidic channel is joined to the first microfluidic channel by at least one T-junction, and e. adjusting said first continuous flow and said second contiguous flow such that aqueous droplets of said hydrophilic solution are generated within said first continuous flow of hydrophobic liquid.
a. providing a hydrophobic liquid in a first reservoir,
b. providing a hydrophilic solution in a second reservoir
c. generating a first continuous flow of said hydrophobic liquid from said first reservoir through at least one inlet, each inlet ending into a first microfluidic channel, wherein the first microfluidic channel enters into a reaction vessel,
d. generating a second continuous flow of said hydrophilic solution from said second reservoir through a second microfluidic channel, wherein the end of said second microfluidic channel is joined to the first microfluidic channel by at least one T-junction, and
e. adjusting said first continuous flow and said second contiguous flow such that aqueous droplets of said hydrophilic solution are generated within said first continuous flow of hydrophobic liquid.
The method of claim 12 , wherein said hydrophilic solution is an aqueous mixture comprising a nucleic acid sample and said hydrophobic liquid comprises an emulsion oil or a mixture of emulsion oils and optionally a surfactant.
The method of claim 12 , wherein the reaction vessel is selected from the group consisting of a single PCR tube, a PCR strip, for example an 8-well strip, and a 96-well plate.
A method for the generation of aqueous droplets in emulsion oil for nucleic acid amplification using a microfluidic chip, device or system according to claim 1 .
Ref US20050202489A1: Claims:
A method for amplifying nucleic acids, which comprises: introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction; creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel; and amplifying the nucleic acids in the reactant aqueous solution droplets.
introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction;
creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel; and
amplifying the nucleic acids in the reactant aqueous solution droplets.
The method of claim 1 , wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.
The method of claim 1 , wherein the operation of amplifying the nucleic acids is performed by one selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and loop-mediated isothermal amplification (LAMP).
The method of claim 3 , wherein the operation of amplifying the nucleic acids is performed by PCR.
The method of claim 4 , wherein the operation of amplifying the nucleic acids is performed by PCR in which the entire surface of a substrate is repeatedly heated and cooled to a predetermined temperature.
The method of claim 4 , wherein the operation of amplifying the nucleic acids is performed by continuous-flow PCR in which a substrate is heated to have at least two different temperature areas and the reactant aqueous solution droplets alternately and repeatedly pass through the different temperature areas.
The method of claim 6 , wherein the substrate is heated to have at least two different temperature areas using a plurality of independently controllable heaters.
The method of claim 4 , wherein the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel further comprises introducing into the reaction vessel at least one aqueous solution of a negative control and a positive control for quantitative assay and a standard sample aqueous solution via respective inlet channels different from the inlet channels for the introduction of the reactant aqueous solution and the fluid, and the operation of creating the reactant aqueous solution droplets further comprises creating a plurality of control aqueous solution droplets and a plurality of standard sample aqueous solution droplets by contacting the at least one aqueous solution of the negative control and the positive control and the standard sample aqueous solution with the fluid in the reaction vessel.
the operation of creating the reactant aqueous solution droplets further comprises creating a plurality of control aqueous solution droplets and a plurality of standard sample aqueous solution droplets by contacting the at least one aqueous solution of the negative control and the positive control and the standard sample aqueous solution with the fluid in the reaction vessel.
The method of claim 8 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the standard sample aqueous solution is adjusted to at least two concentrations and then introduced into the reaction vessel.
The method of claim 4 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, at least two reactant aqueous solutions containing at least two primers are introduced into the reaction vessel via respective inlet channels.
The method of claim 4 , wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, a nucleic acid aqueous solution, a common component containing polymerase and dNTP, and a primer are introduced into respective inlet channels and mixed before being introduced into the reaction vessel to form the reactant aqueous solution.
The method of claim 1 , wherein the reaction vessel comprises two primary reaction channels and a single secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the operation of creating the reactant aqueous solution droplets, and the operation of amplifying the nucleic acids are performed in each of the two primary reaction channels, and wherein after the operation of amplifying the nucleic acids, the method further comprises: combining the reactant aqueous solution droplets in a starting end of the secondary reaction channel to create a plurality of combined reactant aqueous solution droplets in the secondary reaction channel; and amplifying the nucleic acids in the combined reactant aqueous solution droplets.
wherein after the operation of amplifying the nucleic acids, the method further comprises:
combining the reactant aqueous solution droplets in a starting end of the secondary reaction channel to create a plurality of combined reactant aqueous solution droplets in the secondary reaction channel; and
amplifying the nucleic acids in the combined reactant aqueous solution droplets.
The method of claim 1 , wherein the reaction vessel comprises at least one reaction chamber, and wherein after the operation of amplifying the nucleic acids in the reactant aqueous solution droplets, the method further comprises: combining the reactant aqueous solution droplets in the reaction chamber; and amplifying the nucleic acids in the combined reactant aqueous solution droplets.
wherein after the operation of amplifying the nucleic acids in the reactant aqueous solution droplets, the method further comprises:
combining the reactant aqueous solution droplets in the reaction chamber; and
The method of claim 13 , wherein the operation of combining the reactant aqueous solution droplets is carried out by centrifugation.
An apparatus for amplifying nucleic acids, which comprises: a substrate; a reaction vessel formed inside of the substrate; at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel; a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel; and a heating unit installed on the substrate and heating the substrate, the reactant aqueous solution contacting with the fluid to create a plurality of reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and the nucleic acids being amplified in the reactant aqueous solution droplets.
a reaction vessel formed inside of the substrate;
at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel;
a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel; and
a heating unit installed on the substrate and heating the substrate,
the reactant aqueous solution contacting with the fluid to create a plurality of reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and the nucleic acids being amplified in the reactant aqueous solution droplets.
The apparatus of claim 15 , wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.
The apparatus of claim 15 , wherein the substrate has a hydrophobic surface property.
The apparatus of claim 17 , wherein the substrate is made of one material selected from the group consisting of poly(dimethylsiloxane) (PDMS), silicon, silicon dioxide, plastic, and glass.
The apparatus of claim 15 , wherein the substrate comprises a lower substrate and an upper substrate, and the reaction vessel, the first inlet channel, and the second inlet channel are formed between the lower substrate and the upper substrate.
The apparatus of claim 15 , wherein the nucleic acids are amplified in the reactant aqueous solution droplets by one selected from the group consisting of PCR, LCR, SDA, NASBA, TMA, and LAMP.
The apparatus of claim 20 , wherein the nucleic acids are amplified by PCR in the reactant aqueous solution droplets.
The apparatus of claim 21 , wherein the reaction vessel is a serpentine reaction channel, the first inlet channel and the second inlet channel are connected to a staring end of the reaction channel, and an outlet port for releasing the fluid and an amplification product is connected to a terminal end of the reaction channel.
The apparatus of claim 22 , wherein the heating unit is installed at a lower surface of the substrate and comprises at least two heaters for heating the substrate to allow the substrate to have at least two different temperature areas.
The apparatus of claim 22 , wherein the heating unit comprises a plurality of independently controllable heaters installed in parallel at a lower surface of the substrate to heat the substrate so that the substrate has at least two temperature areas.
The apparatus of claim 23 , wherein the reaction channel alternately and repeatedly passes through the temperature areas.
The apparatus of claim 22 , wherein the second inlet channel linearly extends from the starting end of the reaction channel and the first inlet channel is formed perpendicularly to the second inlet channel.
The apparatus of claim 22 , further comprising: a third inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of at least one selected from a negative control aqueous solution and a positive control aqueous solution for quantitative assay into the reaction channel; and a fourth inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of a standard sample aqueous solution for quantitative assay into the reaction channel.
a third inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of at least one selected from a negative control aqueous solution and a positive control aqueous solution for quantitative assay into the reaction channel; and
a fourth inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of a standard sample aqueous solution for quantitative assay into the reaction channel.
The apparatus of claim 27 , wherein the fourth inlet channel is connected to at least two standard sample inlet channels for introduction of standard samples and a distilled water inlet channel for diluting the standard samples to a predetermined concentration.
The apparatus of claim 28 , wherein the fourth inlet channel has a serpentine shape.
The apparatus of claim 22 , wherein the apparatus comprises at least two first inlet channels and the first inlet channels are correspondingly connected to at least two primer inlet channels for introduction of at least two different primers into the first inlet channels.
The apparatus of claim 30 , wherein each of the first inlet channels has a serpentine shape.
The apparatus of claim 22 , wherein the first inlet channel is connected to a primer inlet channel for introduction of a primer into the first inlet channel and a common component inlet channel for introduction of a common component comprising polymerase and dNTP into the first inlet channel.
The apparatus of claim 32 , wherein a portion of a starting end side of the reaction channel has a serpentine shape.
The apparatus of claim 15 , wherein the reaction vessel comprises two serpentine primary reaction channels and a single serpentine secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the first inlet channel and the second inlet channel are connected to starting ends of the two primary reaction channels.
The apparatus of claim 34 , wherein the heating unit comprises a plurality of independently controllable heaters installed at a lower surface of the substrate to provide two temperature areas for each of the two primary reaction channels and the single secondary reaction channel.
The apparatus of claim 15 , wherein the reaction vessel is a reaction chamber, the first inlet channel and the second inlet channel are connected to an end of the reaction chamber, and an outlet channel for releasing the fluid and an amplification product is connected to the other end of the reaction chamber.
The apparatus of claim 36 , wherein the apparatus comprises a plurality of reaction chambers, first inlet channels, second inlet channels, and outlet channels.
The apparatus of claim 36 , wherein the heating unit comprises a single heater installed at a lower surface of the substrate and heating the entire surface of the substrate to a predetermined temperature.