Notice of or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status and Action Summary
This action is in response to the papers filed on December 9, 2025.
Currently, claims 1-33 are pending. Claims 24-33 were withdrawn as being directed to non-elected invention. Claims 1-23 are under examination.
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. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office Action has been withdrawn pursuant to 37 CFR 1.114.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 December 9, 2025 has been entered.
Any objections and rejections not reiterated below are hereby withdrawn.
Priority/Effective Filing Date
The present application, filed June 18, 2021, is a 371 of PCT/US2019/067967, filed December 20, 2019, and claims priority to U.S. Provisional Patent Application No: 62/784,100, filed December 21, 2018.
Drawings
The drawings filed June 18, 2021 are acceptable.
Specification
The use of the terms “AlexaF”, “Alexa Fluro”, and “AF” (Truncations/misspellings of “Alexa Fluor”), “Typhoon”, “New England Biolabs”, “Integrated DNA Technologies” , “Sigma-Aldrich”, “Invitrogen”, “GE Healthcare”, “Corning”, and “TaqMan” which are each a trade name or a mark used in commerce, has been noted in this application. Each term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The listing of references in the specification on page 40-43 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 5-8, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014.
Regarding claims 1-2, Fan et al. teach methods for multiplexed Nucleic Acid detection comprising hybridizing a plurality of oligonucleotide target-specific probes (TSPs) to target nucleic acids, ligating the hybridized TSP, generating amplified TSP sequences, and detecting the amplified TSPs using a plurality of fluorescent probes, wherein a plurality of probes hybridize to binding sites encoded in the TSPs. Fan et al. teach TSPs comprise target binding regions at their 5’ and 3’ ends, a common primer-binding region, and “slots” for fluorescent probe binding sites (Fan et al., Figure 1, reproduced below for convenience).
PNG
media_image1.png
321
498
media_image1.png
Greyscale
Fan et al. teach identifying the target nucleic acids in a particular sample by sequentially hybridizing fluorescently labeled probes to the ligated TSP “slots” wherein the combination of fluorescent signals in the sequential hybridization and imaging steps encodes the identity of each of the plurality of TSPs (Fan et al., paragraphs 0006-0007 and 0040).
Fan et al. do not teach that the fluorescent signals corresponding to the “slot” sequences are multiple copies of a number of fluorescent probe binding regions arranged in particular predetermined copy-number ratios such that the ratio of fluorescence signals corresponding to each of the fluorescent probes encodes the identity of the particular TSP.
However, Gullberg et al. teach methods for detecting ligated padlock probes (i.e. TSPs) comprising hybridizing combinations of fluorescently labeled nucleic acid probes (i.e. FPs) to the amplified TSPs wherein the ratio of measured fluorescent signals is indicative of the identity of each of a plurality of TSPs (Gullberg et al., paragraphs 0050-0056).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods taught by Fan et al. comprising sequential hybridization of fluorescent probes to a plurality of ligated and amplified target specific probes to decode the identity of each of the plurality of TSPs with the teachings of Gullberg et al. that a plurality of ligated and amplified TSPs can be identified by hybridizing a plurality of FPs to the TSPs wherein the FPs bind to the TSPs in predefined ratios such that the resulting fluorescence ratio identifies the TSPs.
The ordinary artisan would have been motivated to modify the methods of Fan et al. comprising sequential hybridization for identification of the TSPs with the ratiometric decoding scheme taught by Gullberg et al. because of the explicit teaching of Gullberg et al., “combinations of target specific [rolling circle probes (i.e. TSPs)] and fluorophore labeled detection oligonucleotides… enables… increasing the obtainable multiplexing-level without the need of a high number of fluorophores or the use of stripping methodologies or other repeated probing (i.e. sequential hybridizations) strategies” (Gullberg et al., paragraph 0050). The ordinary artisan would therefore have expected that the ratiometric fluorescence encoding scheme for TSPs taught by Gullberg et al. would have predictably improved the methods of sequential hybridization and fluorescence detection for decoding TSP identifying-sequences taught by Fan et al.
Regarding claim 5, Fan et al. teach the fluorescent probes (FPs) are nucleic acids (Fan et al., paragraphs 0006-0007 and 0040).
Regarding claim 6, Fan et al. teach amplifying the TSPs by rolling circle amplification, hyperbranched rolling circle amplification, or polymerase chain reaction (Fan et al., paragraph 0029).
Regarding claim 7, Gullberg et al. teach quantitative detection of nucleic acid target sequences in unknown samples (i.e. unidentified nucleic acid target sequences) (Gullberg et al., paragraph 0054).
Regarding claim 8, Fan et al. teach the decoding step(s) comprise hybridization and washing (i.e. removing non-bound nucleic acids) after the ligation step (Fan et al., paragraph 0042).
Regarding claim 23, Gullberg et al. explicitly teach that it is advantageous to increase the number of dyes (i.e. fluorescent probes) used to more than two dyes (i.e. three FPs) (Gullberg et al., paragraph 0054 and 0061).
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014 as applied to claims 1-2, 5-8, and 23 above, and in further view of Kozlowski et al. (2008) (New applications and developments in the use of multiplex ligation-dependent probe amplification; 1 Dec 2008; Electrophoresis 2008, 29, 4627-4636)
The teachings of Fan et al. and Gullberg et al. are presented above.
The combined references do not teach a pair of target specific probes that bind adjacently to the same target. The combined references do not teach that one pair of the probes has a target specific binding region on the 3' while the other of the pair has the target specific binding on the 5'.
However, Kozlowski does teach a pair of target specific probes that bind adjacently to the same target (pg. 4630). Kozlowski teaches that these pair of probes have target specific binding regions, one of which is located at the 3' end, the other at the 5' end (pg. 4630). Kozlowski teaches that the pair of probes both have a common primer-binding region (pg. 4630). This method of probe utilization is more familiarly known in the art as multiplex ligation-dependent probe amplification or MLPA.
It would have been obvious to one of ordinary skill in the art, prior to the filing date of the claimed invention, to have modified the method of Fan et al. and Gullberg et al. with a pair of target specific probes, that bind adjacently to the same target. One pair of the probes having a target specific binding region on the 3' while the other of the pair having the target specific binding on the 5'. Both probes having at least one common primer-binding region with the pair of probes having one or more copies of a fluorescent probe binding region as suggested by Kozlowski.
One of ordinary skill would have been motivated to use the fluorescence ratio of Gullberg for nucleic acid detection because Gullberg teaches that ratiometric detection of ligated probes “enables… increasing the obtainable multiplexing-level without the need of a high number of fluorophores or the use of stripping methodologies or other repeated probing (i.e. sequential hybridizations) strategies” (Gullberg et al., paragraph 0050). They would have been motivated to utilize the probe features of MLPA as it is effective in expression profiling and detection of small mutations/genotyping (pg. 4634 left column).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014 as applied to claims 1-2, 5-8, and 23 above, and further in view of Abravaya et al. (2003) (Molecular beacons as diagnostic tools: Technology and Applications"; 2003; Clinical Chem Lab Med; 41(4):468-474.
The teachings of Fan et al. and Gullberg et al. are presented above.
The combined references do not teach the use of molecular beacons in Multiplex detection of nucleic acid sequences.
However, Abravaya does teach the use of molecular beacons in Multiplex detection of PCR products (abstract).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify the method of Fan et al. and Gullberg et al. by using fluorescent probes that comprise MBs as suggested by Abravaya.
One of ordinary skill in the art would be motivated to use molecular beacons in multiplex detection because Abravaya teaches that they allow for multiplex detection of PCR products in real time in a homogenous assay (abstract).
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014 as applied to claims 1-2, 5-8, and 23 above, and further in view of Johan Baner et al. (1998), (Signal amplification of padlock probes by rolling circle replication; 2 Oct 1998; Nucleic Acids Research, 1998, Vol. 26, No. 22 5073-5078).
Regarding claim 9. The teachings of Fan et al. and Gullberg et al. are presented above.
The combined references do not teach the purification of padlock probes using a spin column.
However, Baner teaches the use of a spin column in the purification process of padlock probes in their use of a Sephadex G-50 (Pg. 5074 right column).
Accordingly, it would have been obvious to one of ordinary skill in the art, prior to the
effective filing date of the claimed invention, to have modified the methods of Fan et al. and Gullberg et al. with the teachings of Baner. One of ordinary skill in the art would have been motivated to utilize a spin column for purification of padlock probes as spin columns are an obvious substitution over known purification methods in the art.
Claims 10, 13-16, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014 as applied to claims 1-2, 5-8, and 23 above, and in further view of Wang et al. (US 2011/0287976 Pub 24 Nov 2011).
The teachings of Fan et al. and Gullberg et al. are presented above.
Fan et al. and Gullberg et al. do not teach a method of SMD detection further comprising “measuring a photon count of each fluorescence color of a single TSS-FP complex comprising the use of cylindrical illumination confocal spectroscopy (CICS)”.
However, Wang does teach the use of an SMD in photon count (Fig. 4) and the use of a cylindrical illumination confocal spectroscopy (CICS) (par. 0017). In claim 22, Wang (US 2011/0287976) describes a microfluidic detection system with a detection region that comprises a detection channel and a confocal fluorescence spectroscope with a confocal aperture, high numeric aperture objective, and a photon detector allowing for single molecule detection.
It would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by using a single molecule detection system coupled with cylindrical illumination confocal spectroscopy to measure photon counts of each fluorescent color as suggested by Wang.
One of ordinary skill in the art would have been motivated to include a CICS SMD into the methods of Fan et al. and Gullberg et al. as these systems are designed to be a highly sensitive and while offering high throughput detection that can be integrated into microfluidics systems (par. 0036).
Regarding claim 13, Gullberg et al. teach a method of detecting target nucleic acids wherein a ratio of measured fluorescent signals generated by hybridization of labeled nucleic acid probes to rolling-circle amplification products of ligated TSPs is indicative of the identity of each of a plurality of TSPs (Gullberg et al., paragraphs 0050-0056).
The combined references of Fan et al. and Gullberg et al. do not teach a method wherein the photon count of each fluorescence color of the TSS-FP complex is measured on CICS.
However, Wang (US 2011/0287976) does teach the use of photon count measuring of fluorescence color. Wang describes single molecule events being measured in bursts utilizing photon counts (par. 0072) with the fluorescent bursts measured using 6xl08 particles/mL, 0.1 mm tetraspec fluorescent beads (par. 0073). Wang also teaches the use of a CICS and its integration into microfluidics systems (par. 0036).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by “measuring the photon count of each fluorescent color of the TSS-FP complex using a CICS” as suggested by Wang. One of ordinary skill in the art would have been motivated to use a CICS to measure photon count for the benefit of a high throughput detection method (par. 0036).
Regarding claim 14. Fan et al. and Gullberg et al. also teach the use of a plurality of ligated TSPs in a nucleic acid amplification mixture and fluorescent probes or FPs (Fan et al., paragraph 0006). Gullberg et al. teach measuring fluorescence ratios to identify nucleic acid sequences (Gullberg et al., paragraph 0055).
The combined references of Fan et al. and Gullberg et al. do not teach loading the ligated TSPs, nucleic acid amplification mixture, and FPs onto an array of discrete receptacles, such that each reaction receptable contains up to one ligated TSP.
However, Wang does teach the use of droplets as reaction receptacles (par. 0026).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by loading the ligated TSPs, nucleic acid amplification mixture, and FPs into droplets, wherein each droplet contains up to one ligated TSP as suggested by Wang. One of skill in the art would have been motivated to utilize droplet receptacles for samples due to their utility in concentrating and orderly processing and analysis of biological target molecules (par. 0026).
Regarding claim 15, Fan et al. teach analyzing samples comprising a plurality of nucleic acid sequences (Fan et al., paragraph 0006).
Regarding claim 16, Fan et al. teach the use of a microfluidics chip (Fan et al., paragraph 0061).
The combined references of Fan et al. and Gullberg et al. do not teach an array of discrete reaction receptacles, wherein the receptacles are droplets.
However, Wang does teach the use of reaction receptacles in the form of a plurality of droplets (par. 0026).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by loading the ligated TSPs, nucleic acid amplification mixture, and FPs into droplets as taught by Wang.
One of ordinary skill in the art would have been motivated to utilize droplet receptacles for separation of samples due to their utility in concentrating and orderly processing and analysis of biological target molecules (par. 0026).
Regarding claim 19, the combined references of Fan et al. and Gullberg et al. do not teach the use of a plurality of droplets having a volume range between 5pL and 1nL.
However, Wang does teach the use of droplets within the range of nano and picoliter sized droplets (par. 0026).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by using droplets that range between 5pL and 1nL as suggested by Wang. One of ordinary skill in the art would have been motivated to utilize droplets within the claimed range as low volume reactors for ease in concentration and use of in sample processing (par. 0026).
Claims 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015, Gullberg et al., US 2014/0194311 A1, published July 10, 2014, and Wang et al. (US 2011/0287976 Pub 24 Nov 2011) as applied to claims 1, 10, 13-16, and 19 above, and further in view of Carlos et al.; (US 2018/0056294 Pub 1 Mar 2018) and Wagner et al.;(WO 2017 /174610 Pub 12 Oct 2017).
The teachings of the combined references are presented above.
Fan et al. and Gullberg et al. do not teach an SMD system that comprises a microfluidic chip comprising a gas permeable silicone material composed of polydimethylsiloxane (PMDS), a transport chamber composed of at least 2 parallel flow channels, each flow channel having a dimension of 5 mm x 0.5 mm.
However, Wang (US 2011/0287976) discloses a SMD comprising a microfluidic chip (par. 0008). Wang teaches that the microfluidic chip comprises a PMDS (Fig. 2c). Wang discloses channels having a width less than about 5 µm and a depth less than about 1 µm (par. 0040).
The combined references do not teach the use of at least 2 parallel flow channels.
However, Wagner does teach the microfluidic chip comprises a transport channel with 2 parallel flow channels (pg. 81 par 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al., Gullberg et al., and Wang by measuring fluorescence of the TSS-TP complexes using a SMD comprising a microfluidic chip comprising a gas permeable silicone material comprising PDMS, a transport chamber composed of at least 2 parallel flow channels, each having a dimension of 5 µm x 0.5 µm as suggested by Wang.
One of ordinary skill in the art would have been motivated to measure fluorescence of the TSS-TP complexes using an SMD comprising a microfluidic chip as SMDs allow the study of molecular properties without the bias of ensemble averaging and the microfluidic chip allows for quantitative detection of low-abundance nucleic acids (Wang, paragraph 0007). They would have been motivated to include a gas permeable silicone membrane of PDMS as it allows for efficient transport of low abundance DNA molecules due to its inert properties (Wang, paragraph 0081).
They would have been motivated to include the 2 parallel flow channels of the dimensions as taught by Wagner, for the benefit of conducting parallel sample processing (Wang par. 0026).
Regarding Claim 11 the combined references do not teach a SMD system comprising a microfluidic chip comprising a filter array at an inlet to reduce flow channel clogging.
However, Carlos does teach the use of a filter near the inlet (par. 0043).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al., Gullberg et al., and Wang by further using a SMD system comprising a microfluidic chip comprising a filter array at the inlet as suggested by Carlos.
Based on the teachings of Carlos the skilled artisan would have been motivated to use a filter near the inlet for the benefit of being able to filter out larger particles or other contaminant which would tend to clog the microfluidic device during operation (Carlos, paragraph 0043).
Regarding claim 12, the teachings of the combined references are presented above.
Fan et al. and Gullberg et al. do not teach the use of nitrogen pressure and gas regulators to drive samples through a microfluidic chip.
However, Wang does teach the use of nitrogen pressure and gas regulators (par. 0074).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. by using a series of precision gas regulators to provide a nitrogen pressure source that pushed the sample through the microfluidic chip as suggested by Wang. One of ordinary skill in the art would have been motivated to use nitrogen pressure in a microfluidic device to maintain a more constant driving force for pervaporation through the device (Wang, paragraph 0074).
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015, Gullberg et al., US 2014/0194311 A1, published July 10, 2014, and Wang et al. (US 2011/0287976 Pub 24 Nov 2011) as applied to claims 1, 10, 13-16, and 19 above, and further in view of Wang et al. (WO 2018/165080, Pub 13 Sep 2018).
The teachings of Fan et al., Gullberg et al., and Wang (US 2011/0287976) are presented above.
Fan et al., Gullberg et al., and Wang et al. (US 2011/0287976) do not teach a chip further comprising a microfluidic flow chamber comprising one or more flow channels and a plurality of picowells with dimensions in the range of 100 pL to 10 nL.
However, Wang et al. (WO 2018/165080) does teach the use of a nL microfluidics system with picowells in contact with flow channels comprising a range of 100 pL to 10 nL (pg. 6-7).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. in view of Wang by using a chip further comprising a microfluidics system with picowells in contact with a flow channel comprising a range of 100pL to 10nL as suggested by Jeff Wang. One of ordinary skill in the art would have been motivated to utilize picowells in contact with flow channels of the claimed range for the benefit of loading and separating nucleic acid for use in amplification reactions.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable Fan et al., US 2015/0368704 A1, published December 24, 2015 in view of Gullberg et al., US 2014/0194311 A1, published July 10, 2014, Wang et al. (US 2011/0287976 Pub 24 Nov 2011) and Wang et al. (WO 2018/165080) as applied to claims 1, 10, 13-17, and 19 above, and further in view of Abravaya (2003), (Molecular beacons as diagnostic tools: Technology and Applications"; 2003; Clinical Chem Lab Med; 41(4):468-474) and Lobofsky et al. (WO 2018/118971 Pub 28 Jun 2018).
Regarding claim 18, the teachings of Fan et al., Gullberg et al., Wang et al (US 2011/0287976), and Wang et al. (WO 2018/165080) are presented above.
The combined references do not teach binding the TSS with the differently labeled molecular beacons.
However, Abravaya does teach the use of molecular beacons in multiplex methods (abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. in view of Wang, and Jeff Wang by binding the TSS with differently labeled MBs as suggested by Abravaya.
One would have been motivated to utilize molecular beacons as they allow multiplex detection of products in real time in homogonous assay formats (abstract).
The combined references do not teach the use of picowells in microfluidics multiplex methods.
However, Lobofsky does teach the use of picowells in microfluidics (par. 0119).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of Fan et al. and Gullberg et al. in view of Wang, Jeff Wang and Abravaya with the use of picowells as taught by Lobofsky.
One would have been motivated to use picowells in multiplex microfluidics as they provide an ideal isolated chamber for low volume fluids for use in biochemical reactions.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015, Gullberg et al., US 2014/0194311 A1, published July 10, 2014, and Wang et al. (US 2011/0287976 Pub 24 Nov 2011) as applied to claims 1, 10, 13-16, and 19 above, and further in view of Revilla et al. (US 2016/0369322, Pub 22 Dec. 2016).
The teachings of Fan et al. and Gullberg et al. are presented above.
Regarding Claim 20, the combined references do not teach a droplet generating module comprising flow channels, microvalves, and flow focusing junction and a droplet measuring module that contains flow channels, microvalves, and a flow constriction channel.
However, Wang (US 2011/0287976) does teach the use of a droplet generator module comprising a flow channel, a focusing droplet generator, and the use of valves in generating droplets (par. 0030 and Fig. 1A).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to have modified the method of Fan et al. and Gullberg et al. by using a droplet generator with flow channel, a focusing droplet generator, and the use of valves in generating droplets as suggested by Wang.
One of ordinary skill in the art would have been motivated to use the droplet generating module with the disclosed features for use in droplet based microfluidics in an SMD as it allows for rapid analysis of molecules trapped within parallel reaction compartments while maintaining an automated and controlled environment (par. 0026)
The combined references do not teach a droplet measurement module.
However, Revilla does teach the use of a droplet measurement module in analyzing fluid samples (Fig. 11). Revilla goes on to describe an analysis module that is capable of measuring droplets in droplet-based reactions in flow cytometry analysis (par. 0080).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to have modified the method of Fan et al., Gullberg et al., and Wang by adding a droplet measurement module as suggested by Revilla.
One of ordinary skill in the art would have been motivated to add a droplet analysis module for the benefit of measuring the concentration of an analyte in a fluid sample (par. 0081)
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015, Gullberg et al., US 2014/0194311 A1, published July 10, 2014, and Wang et al. (US 2011/0287976 Pub 24 Nov 2011) as applied to claims 1, 10, 13-16, and 19 above, as applied to claims 1, 14, and 16 above, and further in view of Wagner et al. (WO 2017 /17 4610 Pub 12 Oct 2017) and Li et al. (Typing of Multiple Single-Nucleotide Polymorphisms by a Microsphere-Based Rolling Circle Amplification Assay; 1 Dec 2007; Anal. Chem. 2007, 79, 9030-9038).
The teachings of Fan et al., Gullberg et al., and Wang et al. are presented above.
Fan et al. and Gullberg et al. do not teach the use of a custom confocal microscope comprising a 488nm laser and a 545 nm laser. Fan et al. and Gullberg et al. do not teach the use of a plurality of dichroic mirrors that combine two laser beams. Fan et al. and Gullberg et al. do not teach a 40x microscope objective to focus the laser beam and collect emitted fluorescence signal from a plurality of droplets.
Fan et al. and Gullberg et al. do not teach the use of a bandpass filter to spectrally separate fluorescence emission signal. Fan et al. and Gullberg et al. do not teach the use of at least two avalanche photodiodes (APD) to collect fluorescence data.
However, Wang (US 2011/0287976) does teach the use of a confocal microscope system measuring fluorescent intensity (abstract). Wang's system teaches the use of a 488nm laser (Fig. 4A), a plurality of dichroic mirrors that combine two laser beams, the use of a microscope objective to focus the laser beam and collect emitted fluorescence (par. 0038). Wang teaches the use of a plurality of dichroic mirrors and a bandpass filter to spectrally separate fluorescence signal (Fig. 4A). Wang also teaches the use of a plurality of APDs to collect fluorescence data (Fig. 4A).
While Gullberg et al. does teach the use of a 40x microscope objective in measuring TSP-FP signals (Gullberg et al., paragraph 0073), the combined references do not teach the use of a 40x microscope objective in microfluidics.
However, Wagner (WO 2017/174610) does teach the use of a 40x microscope objective in microfluidics in their use of the Nikon Super Plan Fluor 40x ELWD (pg. 109, paragraph 3).
Accordingly, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods of Fan et al., Gullberg et al., and Wang et al. with the 40x microscope objective as taught by Wagner.
One of ordinary skill in the art would have been motivated to use a 40x objective in microfluidics as it would have been an obvious substitution over the use of a 40x objective in measuring TSP-FP signals in cells as taught by Gullberg et al. (Gullberg et al., paragraph 0073).
The combined references do not teach the use of a 545 nm laser.
However, Li does teach the use of a of 545 nm laser in their multiplex rolling circle amplification methods (pg. 9032 right column). In the flow cytometer, microspheres were gated and FAM and Cy2 signals in their flow cytometric analysis were measured within the 515-545 nm range.
Accordingly, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods of Fan et al., Gullberg et al., and Wang et al. with the 545nm laser as taught by Li.
One of ordinary skill would have been motivated to include a 545nm laser as it would have been an obvious substitution over the 488 nm laser as taught by Wang (Fig. 4a).
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Fan et al., US 2015/0368704 A1, published December 24, 2015, Gullberg et al., US 2014/0194311 A1, published July 10, 2014, and Wang et al. (US 2011/0287976 Pub 24 Nov 2011) as applied to claims 1, 10, 13-16, and 19 above, as applied to claims 1, 14, and 16 above, and further in view of Li et al. (Typing of Multiple Single-Nucleotide Polymorphisms by a Microsphere Based Rolling Circle Amplification Assay; 1 Dec 2007; Anal. Chem. 2007, 79, 9030-9038) and Revilla et al. (US 2016/0369322, Pub 22 Dec. 2016).
The teachings of the combined references are presented above.
Regarding claim 14. Fan et al. and Gullberg et al. also teach the use of a plurality of ligated TSPs in a nucleic acid amplification mixture and fluorescent probes or FPs (Fan et al., paragraph 0006). Gullberg et al. teach measuring fluorescence ratios to identify nucleic acid sequences (Gullberg et al., paragraph 0055).
Gullberg et al. further teaches incubation and the application of heat in generating target specific sequences by amplification using padlock probes and the use of a surfactant, Tween 20, in the nucleic acid mixture (Gullberg et al., paragraphs 0071-0072 ). Gullberg et al. also teaches the binding of the target specific sequences with fluorescence probes (Gullberg et al., paragraph 0072).
Fan et al. and Gullberg et al. do not teach loading fluid comprising oil, loading droplets on a droplet generation module, separating the mixture into a plurality of droplets with each droplet containing one ligated padlock probe. Fan et al. and Gullberg et al. do not teach the use of a thermal cycler, the use of a droplet measurement module, or the measuring of fluorescence with a confocal microscope system.
However, Wang et al. (US 2011/0287976) does teach the use of oil (par. 0026), a droplet generation module for loading nucleic acid mixtures (par. 0030), and separating this mixture into a plurality of droplets that may contain a single molecule (par. 0026).
Wang further teaches the use of a thermal cycler (par. 0074), and the measuring of fluorescence with a confocal microscope system (par. 0027). Furthermore, Li et al. does teach the use of a of 545 nm laser in their multiplex rolling circle amplification methods (pg. 9032 right column). In the flow cytometer, microspheres were gated and FAM and Cy2 signals in their flow cytometric analysis were measured within the 515-545 nm range.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have to modify the method of Fan et al. and Gullberg et al. by using oil, a droplet generation module for loading nucleic acid mixtures, separating the mixtures into a plurality of droplets that may contain a single molecule, the use of a thermal cycler, and measuring of fluorescence with a confocal microscope system as suggested by Wang et al. (US 2011/0287976) and Li et al.
One of ordinary skill would have been motivated to use oil for microfluidics as oil is known in the art to allow for desirable droplet size. In this regard, relevant to the limitation “plurality of droplets” recited in claim 22, Li et al. teaches microsphere-based rolling circle amplification (RCA) assay (See Title, Abstract), and two padlock probes with the same primer amplification sites were designed to bind to detection probes labeled with distinct fluorescent dyes and target the wild-type and mutant sequences (right column, page 9031). One would have been motivated to include a droplet generation module for loading mixtures and separating mixtures into a plurality of droplets as droplet-based microfluidics are known as an advantageous and complementary technology for single molecule optical platforms that allow for rapid analysis of molecules (par. 0026 of Wang et al. US 2011/0287976).
One of ordinary skill would have been motivated to use a thermal cycler for applying heat in nucleic acid amplification as this is an obvious substitution over the method of heat used by Fan et al. and Gullberg et al. One would have been motivated to utilize a confocal microscope system for measuring fluorescence as these systems are ideally suited as platforms for the detection of rare biomolecules such as nucleic acids (Wang et al., paragraph 0007).
The combined references do not teach a droplet measurement module.
However, Revilla does teach the use of a droplet measurement module in analyzing fluid samples (Fig. 11). Revilla goes on to describe an analysis module that is capable of measuring droplets in droplet-based reactions in flow cytometry analysis (par. 0080).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have to modify the method of Fan et al., Gullberg et al., and Wang et al. by adding a droplet measurement module as suggested by Revilla.
One of ordinary skill in the art would have been motivated to add a droplet analysis module for the benefit of measuring the concentration of an analyte in a fluid sample (Revilla et al., paragraph 0081).
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at 5712723157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682