Notice of Pre-AIA 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 .
DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office Action has been withdrawn pursuant to 37 CFR 1.114.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 November 1, 2024 has been entered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Status of Claims
Claims 31-33, 35, 36, 38-40, 45, 47-49 and 51-56 are currently pending in the instant application. Claims 31, 33, 35, 38-40, 45 and 47-49 have been amended by Applicants’ amendment filed 11-01-2024. Claim 50 has been canceled by Applicants’ amendment filed 11-01-2024. Claims 51-56 have been added by Applicants’ amendment filed 11-01-2024.
Therefore, claims 31-33, 35, 36, 38-40, 45, 47-49 and 51-56 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed January 12, 2021 is a CON of US Patent Application 17/146,974, filed January 12, 2021, which claims the benefit of US Provisional Patent 62/960,283, filed January 13, 2020.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on November 1, 2024; January 14, 2025; and April 4, 2025 have been considered. Initialed copies of the IDSs accompany this Office Action.
Withdrawn Objections/Rejections
Applicants’ amendment and arguments filed November 1, 2024 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or objection not specifically addressed below are herein withdrawn.
Claim Rejections - 35 USC § 103
The rejection of claims 31-33, 35, 36, 38-40, 45 and 47-49 is withdrawn under 35 U.S.C. 103 as being unpatentable over Abate et al. (US20200261879; effective filing date at least as of September 28, 2018 based on PCT/US2018/053598; of record) in view of Tawfik et al. (US Patent Application No. 20080004436, published January 3, 2008; of record) as evidenced by DiagnoCine (DC DiagnoCine, 2023, 1-11; of record); and Thomas Scientific (ACTGene, 2023, 1-6; of record); and Link et al. (US Patent No. 11596908, issued March 7, 2023; also published as US20180353913, published December 13, 2018; effective filing date July 18, 2008; of record); and Johnson (Material Methods, 2013, 3, 163; of record); and Schuch et al. (Sensors, 2007, 7, 2499-2509; of record).
Based on the amendments to the claims, Abate and Tawfik are not the strongest combination of references.
In view of the withdrawn reference, Applicant’s arguments are rendered moot.
Maintained Objections/Rejections
Claim Rejections - 35 USC § 112(b)
The rejection of claims 31-33, 35, 36, 38-40, 45 and 47-49 is maintained, and claims 51-56 is newly rejected, under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Claims 31, 45, 47, 48 and 56 are indefinite for the recitation of the term “the cell” such as recited in claim 31, lines 4 and 5. There is insufficient antecedent basis for the term “the cell” in the claim because claim 31, line 3 recites the term “at least one cell.” The Examiner suggests that Applicant amend the claims to recite, for example, “wherein at least one of the aqueous droplets contains at least one cell.”
Claims 31, 45, 48 and 56 are indefinite for the recitation of the term “the aqueous droplet” such as recited in claim 31, line 5 because it is unclear whether there is a single aqueous droplet or a plurality of aqueous droplets. For example, claim 31, lines 2 and 4 recite the terms “into aqueous droplets” and “at least one of the aqueous droplets,” while also reciting the term “the aqueous droplet” and, thus, the metes and bounds of the claim cannot be determined. The Examiner suggests that Applicant amend the claims to recite, for example, “at least one of the aqueous droplets,” “each aqueous droplet in the plurality of aqueous droplets,” or “the plurality of aqueous droplets.”
Claims 33, 48 and 55 are indefinite for the recitation of the term “the delivering step” such as recited in claim 33, line 2. There is insufficient antecedent basis for the term “the delivering step” in the claim because claim 31, line 5 recites the term “delivering a micellized lysis agent.” Moreover, instant claim 31 does not recite a “delivering step.”
Claim 35 is indefinite for the recitation of the term “surfactant” in claim 35, line 2 because it is unclear whether the term “surfactant” encompasses the same compounds, mixtures, and/or molecules such as the “micellized lysis agent,” “fluorosurfactant,” and/or “detergent,” such as recited in claims 31, 33, 39, 49 and 51-55 and as evidenced by ThermoFisher (pgs. 1-6) and, thus, the metes and bounds of the claim cannot be determined.
Claim 35 is indefinite for the recitation of the term “a plurality of the aqueous droplets” in claim 35, line 3 because it is unclear whether the “a plurality of the aqueous droplets” recited in claim 35 is the same as (or is different from) “the aqueous droplets” and/or “the at least one of the aqueous droplets” recited in claim 31, lines 2 and 4 and, thus, the metes and bounds of the claim cannot be determined.
Claim 39 is indefinite for the recitation of the term “detergent” in claim 39, line 2 because claim 39 depends from instant claim 31, wherein it is unclear whether the micellized lysis agent comprising a “detergent” as recited in claim 39 is referring to the “surfactant” recited in claim 31; or whether the micellized lysis agent comprises both a detergent and a surfactant or a heat activated enzyme. Moreover, it is unclear whether the term “detergent” encompasses the all of the same compounds and/or molecules as encompassed by the terms “surfactant,” “micellized lysis agent” and “fluorosurfactant” such as recited, for example, in claims 31, 33, 35 and 36and as evidenced by ThermoFisher (pgs. 1-6); or whether there is some difference between the terms and, thus, the metes and bounds of the claim cannot be determined.
Claims 39 and 54 are indefinite for the recitation of the term “heat activated enzyme” such as recited in claim 39, line 2 because it is unclear which enzymes are encompassed by the term “heat activated enzymes” including whether an enzyme that is inactive at 0oC, but having some activity at 5oC, is considered to be a “heat activated enzyme;” whether the term refers to enzymes having their greatest activity at room temperature or 70oC, whether the enzymes have an optimal temperature or an optimal temperature range; and/or whether the term refers to some other type of “heat-activated enzyme” and, thus, the metes and bounds of the claim cannot be determined.
Claim 47 is indefinite for the recitation of the term “forming cDNA, forming amplicons from the cDNA, sequencing the amplicons to generate an expression profile, and analyzing the expression profile” in claim 47, lines 1-5 because claim 47 depends from claim 31, wherein claim 31 is directed to a “method of preparing a sample” and not to a method of sequencing a sample, or a method of identifying a sample.
It is noted that claim 31 does not recite the presence or isolation of RNA, that reactions are carried out within or outside of the aqueous droplet; it does not indicate whether cDNA is formed in the body of a subject or by chemical reaction on a lab bench; it does not recite the presence of amplicons; devices for sequencing and analysis; there is no gene expression; no indication of what is being expressed (e.g., a protein ?) such that an expression profile can be produced; cell lysis; the presence of reagents necessary to carry out chemical reactions such as reverse transcription and cDNA synthesis (e.g., reverse transcriptase, dNTPs, solvents, surfactants, inteins, etc.); method or devices for analyzing and/or fragmentation; the production of an expression profile, etc. such that instant claim 47 appears to be omitting essential steps and, thus, the metes and bounds of the claim cannot be determined.
Claim 48 is indefinite for the recitation of the term “sequencing nucleic acids from the cells in the aqueous droplet after the delivering step” in claim 48, lines 1-3 because methods steps and/or reagents required to provide a sequencing library are omitted. Therefore, it is unclear how “sequencing of nucleic acids” can be carried out on the aqueous droplets given that cell lysis, isolation into fluid partitions, and/or amplification have not occurred, there are no amplicons, cDNA; NGS adaptors, primers, barcodes, etc. present for sequencing to be carried out and, thus, the metes and bounds of the claim cannot be determined.
Claims 51-53 are indefinite for the recitation of the terms “Sakosyl,” “Capstone FS-61,” “CTAB,”NP-40,” “SB3-10,” “Brij 35,” “CHAPS,” “CHAPSO” etc. for the recitation of trademarks or trade names. It is noted that where a trademark or trade name (research designation) is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark or trade name is used to identify/describe either a detergent or a heat activated enzyme and, accordingly, the identification/descriptions are indefinite. For the sake of compact prosecution the “Sakosyl,” “Capstone FS-61,” and “CTAB” have been interpreted as any type of surfactants/detergents.
Claim 55 is indefinite for the recitation of the term “oil phase” in claim 55, line 2 because claim 55 depends from claim 31, wherein claim 31 recites a sample in aqueous droplets, but it does not recite the presence of an “immiscible phase” such as an “oil phase.” Moreover, because micelles do not have an “oil phase” it is unclear what the surfactant of claim 55 is referring to and, thus, the metes and bounds of the claim cannot be determined.
Claim 56 is indefinite for the recitation of the term “forming cDNA from mRNA” in claim 56, lines 1 because claim 56 depends from claim 31, wherein claim 31 does not recite steps and/or reagents leading to the formation of cDNA from mRNA including, for example, cell lysis, the isolation of mRNA, whether reactions are carried out within the aqueous droplet or outside of the aqueous droplet; the location of cDNA synthesis is unclear (e.g., within the body of a subject or by chemical reaction on a lab bench); reagents for reverse transcription and cDNA synthesis (e.g., reverse transcriptase, dNTPs, solvents, surfactants, inteins, etc.), etc. and, thus, the metes and bounds of the claim cannot be determined.
Claims 32, 36, 38, 40 and 49 are indefinite insofar as they ultimately depend from claim 31.
Claim Rejections - 35 USC § 112(d)
The rejection of claim 47 is maintained, and claims 48, 55 and 56 are newly rejected, under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 47 recites (in part): “forming cDNA; forming amplicons from the cDNA; sequencing the amplicons to generate an expression profile; and analyzing the expression profile of the cell” in claim 47, lines 2-5 because claim 47 depends from instant claim 31, wherein claim 31 does not recite the presence and isolation of mRNA; the presence of devices and/or methods of fragmentation, sequencing, analysis, measuring an expression of something; and/or for producing an expression profile; and reagents necessary to carry out chemical reactions such as reverse transcription and cDNA synthesis (e.g., reverse transcriptase, dNTPs, solvents, surfactants, inteins, etc.). Thus, claim 47 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 48 recites (in part): “sequencing nucleic acids from the cells in the aqueous droplet after the delivering step” in lines 1-3 because claim 48 depends from instant claim 31, wherein claims 31 and 48 have omitted process steps and/or reagents taught in the as-filed Specification that would lead to sequencing including, for example, there is no cell lysis, no nucleic acid isolation and/or amplification; and there are no amplicons, cDNA, NGS adaptors, primers, barcodes, sequencing device, etc. recited such that sequencing to be carried out. Thus, claim 48 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 55 recites (in part): “further comprising prior to the delivering step, using a surfactant to micellize the lysis agent into an oil phase” in claim 55, lines 1-2 because claim 55 depends from instant claim 31, wherein claim 31 does not recite the presence of an oil phase or an immiscible liquid. Claim 31 recites at least one aqueous droplet. Thus, claim 55 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 56 recites (in part): “further comprising: extracting mRNA from the cell in the aqueous droplet and forming cDNA from the mRNA” in claim 56, lines 1-3 because claim 56 depends from claim 31, wherein claim 31 does not recite the presence or isolation of mRNA, that reactions are carried out within or outside of the aqueous droplet; it does not indicate whether cDNA is formed in the body of a subject or by chemical reaction on a lab bench; reagents for cell lysis; the presence of reagents necessary to carry out reverse transcription and cDNA synthesis (e.g., reverse transcriptase, dNTPs, solvents, surfactants, inteins, etc.), etc. Thus, claim 56 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 103
The rejection of claims 31-33, 35, 36, 38-40, 45 and 47-49 is maintained, and claims 51-56 are newly rejected, under 35 U.S.C. 103 as being unpatentable over Delaney et al. (US2020188920; published June 18, 2020; effective filing date June 11, 2018; of record) in view of Abate et al. (US20200261879; effective filing date at least as of Sept. 28, 2018 based on PCT/US2018/053598; of record) as evidenced by Johnson (Material Methods, 2013, 3, 163); and Schuch et al. (Sensors, 2007, 7, 2499-2509; of record); and Chae et al. (Molecular Biosystems, 2010, 6, 89-94); and Abate et al. (hereinafter “Abate ‘507”) (US Patent No. 10434507, issued October 8, 2019; effective filing date October 22, 2014); and Chowdhury et al. (Nature Communications, 2019, article 4546, 1-10).
Regarding claims 31-33, 35, 36, 38-40, 45, 47-49 and 51-56, Delaney et al. teach methods for forming emulsions, comprising: bringing a first fluid phase in contact with a second fluid phase that is immiscible with the first fluid phase to generate the emulsion comprising the plurality of droplets, wherein the plurality of droplets can comprise (i) the first fluid phase or the second fluid phase, (ii) a first surfactant at an interface between the first fluid phase and the second fluid phase, and (iii) a second surfactant that is different than the first surfactant, such that subsequent to generating the emulsion, collect or direct the plurality of droplets along a channel, wherein at most 5% of the plurality of droplets coalesce (interpreted as simultaneously forming an emulsion of water and an immiscible fluid; a surfactant; and merging emulsion droplets, claims 31 and 35) (Abstract). Delaney et al. teach surfactant systems for stabilizing droplets of water and hydrocarbon oil or organic solvents in a continuous fluorophilic phase are needed including water-in-fluorocarbon emulsions (interpreted as an aqueous droplet or a droplet comprising a cell, claim 31) (paragraphs [0004]; and [0035]). Delaney et al. teach in Figure 1, a droplet generator comprising a microfluidic channel structure for partitioning individual or small groups of cells (interpreted as a first emulsion comprising cells; partitioning; and one or no cells, claims 31 and 43) (paragraph [0013]; and Figure 1). Delaney et al. teach that emulsions can comprise a hydrocarbon oil phase and an aqueous phase; and that fluorocarbon oils can be immiscible with both the hydrocarbon oils and the aqueous phase; as well as, stabilization of emulsions in fluorocarbon oil can require the addition of appropriate surfactants such as a fluoro-surfactant (interpreted as comprising a fluorosurfactant, claims 31, 33 and 36) (paragraphs [0004]-[0005]). Delaney et al. teach that the term "droplet" generally refers to an isolated aqueous or lipophilic phase within a continuous phase having any shapes (interpreted as an aqueous droplet or a droplet comprising a cell, claim 31) (paragraph [0036]). Delaney et al. teach in droplets in an emulsion, allocating individual cells to discrete partitions can generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams, such that by providing the aqueous cell-containing stream at a certain concentration of cells, the occupancy of the resulting partitions (e.g., number of cells per partition) can be controlled (interpreted as a first emulsion comprising cells; and one or no cells on average, claims 31 and 43) (paragraph [0077], lines 1-9). Delaney et al. teach that Figure 9 illustrates an emulsion droplet/micelle 900 that can comprise lysis agent n-dodecyl-b-D-maltoside (DBDM) 902 inside its aqueous phase and a plurality of tri-block surfactants 904 including 904A, 904B and 904C forming a barrier at the interface between the fluorocarbon oil phase and the aqueous phase during emulsification (interpreted as a first and second emulsion comprising micelles and a lytic agent; and using a surfactant to micellize the lysis agent, claims 31, 39 and 55) (paragraph [0145], lines 4-9; and Figure 9). Delaney et al. also teach in Figure 9, that an emulsion droplet/micelle 900 can comprise a lysis agent DBDM 902, and a plurality of new di-block surfactants 910A-C, forming a barrier at the interface between the fluorocarbon oil phase and the aqueous phase during emulsification, wherein each new di-block surfactant 910 can comprise one fluorophilic tail 912 and one hydrophilic head groups 914 (interpreted as micellized lysis agents delivered to a cell; and prepared with a fluorosurfactant; and DBDM as a detergent, claims 31, 33, 36, 39 and 55) (paragraph [0146], lines 1-9). Figure 9 is shown below:
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Delaney et al. teach NGS sequencing platforms and technologies such as the Illumina Genome Analyzer, the Roch (454) Genome Sequencer, the Life Technologies SOLiD platform, and real-time sequences such as by Pacific Biosciences (interpreted as sequencing, claims 47 and 48) (paragraph [0044], lines 1-5). Delaney et al. teach that the nucleic acids derived from the cells can include DNA, or RNA, including, e.g., mRNA, total RNA, or the like, that can be processed to produce cDNA for sequencing (interpreted as extracting mRNA from a cell; and forming cDNA for sequencing, claims 47 and 56) (paragraph [0044], lines 13-17). Delaney et al. teach that surface-mediated coalescence can occur with tri-block surfactants and the lysis agents such as DBDM in the presence of a roughened surface (interpreted as merging the first emulsion and second emulsion comprising cells and a lytic agent, claim 31) {paragraph [0146], lines 30-33). Delaney et al. teach that a plurality of droplets can be subjected to flow or directed along a channel with the aid of a flow system, wherein fluid can be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, and the like (interpreting centrifugal force as preparing by vortexing at the require rpm; and agitating and mixing, claims 31, 38 and 40) (paragraph [0104]). Delaney et al. teach that different lysis agents can be used including glycosides and maltosides with alkyl chains (interpreted as encompassing surfactants octyl glucosides or octyl thioglucoside, claim 52) (paragraph [0123]). Delaney et al. teach that the fluidic droplets can be of uniform size (interpreted as monodisperse aqueous droplets, claim 32) (paragraph [0058], lines 31-32). Delaney et al. teach that emulsions are imaged under the microscope and the coalescence levels are determined using a software developed to identify droplets within an image and bin each droplet according to size (interpreted as monodisperse aqueous droplets, claim 32) (paragraph [0166], lines 16-20). Delaney et al. teach that the method are useful for a variety of different molecular biology applications including nucleic acid sequencing, protein sequencing, detecting gene expression, quantifying gene expression, epigenetic applications, and single cell analysis of genomic or expressed markers; as well as, for the identification, detection, diagnosis, treatment, staging of, or risk prediction of various genetic and non-genetic disease and disorders including cancer (interpreted as analyzing expression profiles; and sequencing, claims 47 and 48) (paragraph [0074]). Delany et al. teach that the cells can be partitioned along with lysis reagents in order to release the contents of the cells within the partition, wherein the lysis agents can be contacted with the cell suspension concurrently with, or immediately prior to the introduction of the cells into the partitioning junction/droplet generation zone, such that Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes; as well as, other commercially available lysis enzymes, wherein the other lysis agents can additionally or alternatively be co-partitioned with the cells to cause the release of the cell's contents into the partitions including surfactant based lysis solutions can be used to lyse cells, wherein these lysis surfactants can interfere with stable emulsions; and that the lysis solutions can include non-ionic surfactants such as, for example, Triton X-100 and Tween 20; such that the lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS); and that electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of cells that may be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a desired size, following cellular disruption (interpreted as lysis agents delivered to cells including cells in a droplet including Triton X-100, SDS, sarkosyl, Tween; heat activated enzymes; micellized lysis agents created prior to delivery; and detergent types including ionic, non-ionic, and zwitterionic types, claims 31, 33, 39, 49 and 51-54) (paragraph [0094]). Delaney et al. teach detergent-based cell lysis can be a milder and easier alternative to physical disruption of cell membranes, wherein it can be used in conjunction with homogenization and mechanical grinding; and that detergents can break the lipid barrier surrounding cells by solubilizing proteins and disrupting lipid:lipid, protein:protein and protein:lipid interactions, such that detergents can be categorized by the nature of the head group as either ionic (cationic or anionic), nonionic or zwitterionic, wherein lysis agents can include, but not be limited to, hemi-fluorinated maltosides, zwitterionic agents (comprising both negative and positive charges) and tripod amphiphile (See; Chae, “Tripod Amphiphiles for Membrane Protein Manipulation,” 2010) (interpreted as encompassing detergents including CHAPS, ASB-14, NP-40, etc., claims 39 and 51-55) (paragraph [0126]), wherein it is known that Tripod amphiphile design includes classical detergents, as exemplified by n-dodecyl--D-maltoside (DDM) and lauryl dimethylamine-N-oxide (LDAO) 3-[(3-cholamindopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as evidenced by Chae et al. (pg. 90, col 1; last partial paragraph). Delaney et al. teach methods, systems and compositions useful in the processing of sample materials, for example, nucleic acids samples, through the controlled delivery of reagents to subsets of sample components, followed by analysis of those sample components employing, in part, the delivered reagents (interpreted as delivering reagents to cells including lysis agents, claim 31) (paragraph [0049], lines 1-6). Delaney et al. teach that beads can serve as a carrier for reagents that are to be delivered, wherein a variety of different reagents or reagent types can be associated with the beads, when delivering such reagents to a partition, wherein non-limiting examples of such reagents delivered include, e.g., enzymes, polypeptides, antibodies or antibody fragments, labeling reagents, e.g., dyes, fluorophores, chromophores, etc., nucleic acids, polynucleotides, oligonucleotides ((interpreted as delivering reagents such as lysis agents, claim 31) (paragraph [0050]). Delaney et al. teach that microcapsules are used to deliver additional reagents to a partition, wherein surface-mediated coalescence in the presence of DBDM can occur (interpreted as delivering reagents such as lysis agents, claim 31) (paragraphs [0082], lines 1-2; and [0146]).
Delaney et al. do not specifically exemplify creating micellized lysis agent prior to the delivery step (claims 33, in part).
Regarding claims 33 (in part), Abate et al. teach a "particle-templated emulsification” (PTE)" method for generating a monodisperse emulsion including monodisperse single-emulsion droplets, multiple-emulsion droplets, and Giant Unilamellar Vesicles (GUVs) that encapsulates target particles of interest, such as cells, without requiring the use of a microfluidic device (interpreted as comprising cells; a first and second emulsion; and partitioning into micelles, claim 31) (Abstract; and paragraph [0004]). Abate et al. teach methods for generating a monodisperse emulsion include combining a plurality of monodisperse template particles with a first fluid to provide a first mixture, wherein the first fluid includes a plurality of target particles; combining the first mixture with a second fluid to provide a second mixture, wherein the second fluid is immiscible with the first fluid; and shearing the second mixture such that a plurality of the monodisperse template particles are encapsulated in a plurality of monodisperse droplets in the second fluid, thereby providing a plurality of monodisperse droplets including the first fluid, one of the monodisperse template particles, and one of the plurality of target particles in a monodisperse emulsion (interpreting shearing and vortexing as mixing; monodisperse droplets; interpreted as preparing a first emulsion and a second emulsion and micelles, claims 31, 32 and 40) (paragraph [0005]). Abate et al. teach that target particles can include a heterogeneous population of cells, viruses and/or nucleic acids, which can be diluted to encapsulate a controlled number of cells, viruses, and/or nucleic acids in monodisperse droplets; as well as, co-encapsulating nucleic acid synthesis reagents including amplification reagents and/or PCR reagents along with one or more target particles to facilitate downstream detection, sorting and/or analysis (interpreted as one cell within droplets, claims 31 and 43) (paragraph [0006]). Abate et al. teach contacting lysis agents with the cell suspension concurrently with, or immediately prior to the introduction of cells into the partitioning junction/droplet generation zone; and/or when the cells have been suspended in the aqueous fluid, or pre-encapsulated, prior to the partitioning process (interpreted as micellized lysis agents created prior to the delivering step, claim 33) (paragraphs [0094]; and [0097]). Abate et al. teach in Figure 3, vortexing emulsification, wherein Panel E depicts the addition of oil before vortexing; and generating emulsions by vortexing (interpreted as vortexing; and interpreting vortexing as mixing, claims 31 and 38) (paragraph [0029]; and Figure 3). Figure 3 is shown below:
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Abate et al. teach that mixing can partition the sample and bead mixture into a plurality of fluidic droplets (e.g., aqueous droplets within a water-in-oil emulsion), in which at least a subset of the droplets may encapsulate a barcoded bead such as a gel bead (interpreted as partitioning a sample in an aqueous droplet, claim 31) (paragraph [0063]). Abate et al. teach a microfluidic device that can be used to combine sample with beads (e.g., a set of barcoded beads); as well as, an agent capable of degrading the beads (interpreted as delivering a lysis agent to a sample in an aqueous droplet, claim 31) (paragraph [0062]). Abate et al. teach in Figure 5, images of droplets prepared under different PTE conditions as described in Example 2, wherein Panel C depicts shearing a second mixture, wherein the monodisperse particles are encapsulated in monodisperse droplets prepared by vortexing PAA particles with 2% Tween 20; 0.5% Triton in HFE oil with 2% fluorosurfactant including a second fluid to provide monodisperse droplets and one of the target particles, wherein target particles include cells (interpreting vortexing Tween 20, Triton in HFE oil, and fluorosurfactants as micellized lysis agents that are delivered to a cell via a fluorosurfactant including cells in droplets because vortexing forms emulsion droplets; lysis agent is a detergent; mixing by vortexing at 200-700 rpm; creating agent prior to delivering; droplets surrounded by an immiscible fluid; fluorosurfactant; and lysis agent is ionic type, non-ionic type or zwitterionic type; and interpreting target particles to include cells, claims 31, 33, 35, 36, 38-40, 45 and 49) (paragraphs [0028]; [0031]; and [0338]); wherein it is known that surfactants can form micelles above the critical micelle concentration, and the formation of micelles is expected in all cases of higher surfactant concentrations in microreactors as evidenced by Schuch et al. (pg. 2500, last partial paragraph; and pg. 2501, first partial paragraph); and where it is known that carrier phases with surfactant can undergo micellar transport allowing compounds to be transported into or out of droplets as evidenced by Abate ‘507 (col 26, lines 20-23). Abate et al. teach that Panel D depicts a fluorescent image of a formed liposome with a fluorescent lipid in the oil phase produced by adding oil with stabilizing surfactant including droplets prepared by mixing PAA particles with 0.5% Triton with FC-40 oil and 5% fluorosurfactant and vortexing, wherein calcine green stained cells are encapsulated into droplets before lysing (interpreting vortexing Tween 20, Triton in HFE oil, and fluorosurfactants as micellized lysis agents that are delivered to a cell via a fluorosurfactant including cells in droplets because vortexing forms emulsion droplets; lysis agent is a detergent; mixing by vortexing at 200-700 rpm; creating agent prior to delivering; droplets surrounded by an immiscible fluid; fluorosurfactant; and lysis agent is ionic type, non-ionic type or zwitterionic type; and interpreting target particles to include cells, claims 31, 33, 35, 36, 38-40, 45 and 49) (paragraph [0029]; [0031]; [0039]; and [0041]), wherein it is known that detergents include ionic, non-ionic and zwitterionic chemicals such as SDS, Triton X-100, sarkosyl, deoxycholate, DDM, digitonin, tween 20, tween 80, non-idet, CHAPS, Igepal CA-630 and urea, which are used for cell lysis as evidenced by Johnson (Abstract; pg. 163, first full paragraph; first full paragraph; and Table 1; and pg. 167, last partial paragraph). Abate et al. teach in Figures 14A-C a diagram to perform high throughput scRNA-seq through PTE, wherein Figure 14A depicts Drop-seq beads being encapsulated into hydrogels, wherein cells, proteinase K and hybridization buffer are mixed; Figure 14B depicts vortexing the mixture for emulsification; and Figure 14C depicts recovering the Drop-seq beads and RT sequencing, followed by data analysis (paragraph [0040]; and Figures 14A-C). Figures 14A-C is shown below:
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Abate et al. teach that excess aqueous phase was removed with a micropipette, particles and HFE-7500 oil supplemented with 2% (w/w) PEG-PFPE amphiphilic block copolymer surfactant (008 Fluoro-surfactant, Ran Technologies) were mixed well in an Eppendorf Tube, and the mixture was agitated at 2300 rpm for 30 seconds with a vortexer (interpreted as forming micelles prior to delivering, claims 31 and 55) (paragraph [0319]), wherein it is known that the non-ionic tri-block copolymer fluorosurfactant, PEG-PFPE2 made of poly(ethylene glycol) and perfluoropolyethers (PFPE), is widely used to stabilize the emulsion droplets within fluorinated oil, such that surfactants with a tri-block structure can efficiently form micelles and bilayer vesicles as evidenced by Chowdhury et al. (pg. 2, col 1, first full paragraph). Abate et al. teach RNAseq, wherein the beads, cells and RT mix are combined and PETE is performed as described herein to encapsulate single cell in droplets with reverse transcription reaction mix, wherein the emulsion is exposed to UV light followed by heating up to 50° C to perform the reverse transcription reaction, such that during the process, single strand DNA on beads is released to function as RT primers and the RNA from the cell is released to function as templates; and the emulsion is then broken and cDNA is recovered followed by standard in vitro transcription and library preparation for next generation sequencing to collect the data of single cell gene expression profile analysis (interpreted as droplets comprising one cell, claim 31 and 40) (paragraph [0354], lines 9-21).
It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of generating monodisperse emulsions that encapsulate target particles of interest as exemplified by Abate et al., it would have been prima facie obvious to one having ordinary skill before the effective filing date of the claimed invention to modify the method of forming emulsion droplets/micelles including micellized lysis agents including glycosides, maltosides, detergents, and/or surfactants including ionic, non-ionic and/or zwitterionic lysis agents as disclosed by Delaney et al. to include the methods of forming emulsions including contacting lysis agents with the cell suspension concurrently with, or immediately prior to the introduction of cells into the partitioning junction/droplet generation zone; and/or when the cells have been suspended in the aqueous fluid, or pre-encapsulated, prior to the partitioning process as taught by Abate et al. with a reasonable expectation of success in providing an improved, cost-effective, and efficient method for the generation and/or stabilization of droplets including emulsions and micelles; as well as, an improved method for the encapsulation of target particles including cells with and/or without the use of a microfluidic device. Moreover, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of preparing emulsion droplet/micelles using an aqueous phase and immiscible oils such as silicon oil, mineral oil, vegetable oil, and fluorinated oils including HFE-7100 and HFE-7500; and surfactants such as Ran or ionic Krytox as exemplified by Delaney et al. to include one or more surfactants such as PEG-PFPE, PEG-Krytox, Krytox 157 FSH, Triton X-100, and IGEPAL as taught by Abate et al. with a reasonable expectation of success in generating micelles, emulsion droplet/micelles, and/or monodisperse emulsion droplets useful for encapsulating and/or transferring biological samples including cells; lytic agents and/or barcoded beads.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly rejected under 35 USC §103 as obvious over the art.
Response to Arguments
Applicant’s arguments filed November 1, 2024 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) Abate as evidenced by Schuch and Johnson fails to teach delivering a micellized lysis agent to the cell as recited in claim 31 (Applicant Remarks, pg. 14; second full paragraph, lines 1-2); and (b) the Examiner acknowledges that Delaney fails to teach delivering a micellized lysis agent (Applicant Remarks, pg. 14, second full paragraph, line 3).
Regarding (a), it is noted that although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26USPQ2d 1057 (Fed. Cir. 1993). Moreover, as noted in MPEP 2112.01(I),
where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Additionally, MPEP 2123(I) states:
"The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill the art, including nonpreferred embodiments. See In re Heck, 699 F.2d 1331, 1332-33,216 USPQ 1038, 1039 (Fed. Cir. 1983); In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275,277 (CCPA 1968); Merck & Co. v. Biocraft Laboratories, 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); and Upsher-Smith Labs. v. Pamlab, LLC, 412 F.3d 1319, 1323, 75 USPQ2d 1213, 1215 (Fed. Cir. 2005) (underline added).
Moreover, none of the references has to teach each and every claim limitation. If they did, this would have been anticipation and not an obviousness-type rejection. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Applicant’s assertion that Abate as evidenced by Schuch and Johnson fails to teach delivering a micellized lysis agent to the cell as recited in claim 31, is not found persuasive. As an initial matter, the Examiner notes that the claims are rejected over a combination of references, in this case Delaney and Abate. Applicant has failed to address the teachings of Delaney and/or the combined teachings of Delaney and Abate. The Examiner asserts that the combined references teach all of the limitations of the claims. For example -
Delaney et al. teach:
A droplet generator comprising a microfluidic channel structure for partitioning individual or small groups of cells (interpreted as a droplet or aqueous droplet containing a cell).
That cells can be partitioned along with lysis reagents in order to release the contents of the cells within the partition, wherein the lysis agents can be contacted with the cell suspension concurrently with, or immediately prior to the introduction of the cells into the partitioning junction/droplet generation zone.
Figure 9 shows an emulsion droplet/micelle 900 can comprise a lysis agent DBDM 902 inside its aqueous phase, wherein Figure 9 is shown below:
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where it is known that carrier phases with surfactant can undergo micellar transport allowing compounds to be transported into or out of droplets as evidenced by Abate ‘507.
Methods, systems and compositions useful in the processing of sample materials, for example, nucleic acids samples, through the controlled delivery of reagents to subsets of sample components, followed by analysis of those sample components employing, in part, the delivered reagents (interpreted as delivering reagents to cells including lysis agents).
Examples of lysis agents include:
Bioactive reagents, such as lysis enzymes that are used for lysis of different cell types (e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes);
Commercially available lysis enzymes.
Surfactant-based lysis solutions can be used to lyse cells.
Non-ionic surfactants such as, for example, Triton.X-100 and Tween 20.
Ionic surfactants such as sarkosyl and sodium dodecyl sulfate (SDS).
That electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of cells that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a desired size, following cellular disruption.
(interpreted as delivering lysis agents to the cell including to the cell in a droplet, wherein lysis agents include Triton X-100, SDS, sarkosyl, Tween, etc.).
Abate et al. teach:
Figure 13, Panel D depicts a fluorescent image of a formed liposome with additional fluorescent lipid in the oil phase (interpreted as an emulsion of claim 35).
Excess aqueous phase was removed with a micropipette, particles and HFE-7500 oil supplemented with 2% (w/w) PEG-PFPE amphiphilic block copolymer surfactant (008 Fluoro-surfactant, Ran Technologies) were mixed well in an Eppendorf Tube, and the mixture was agitated at 2300 rpm for 30 seconds with a vortexer (interpreted as forming micelles);
wherein it is known that the non-ionic tri-block copolymer fluorosurfactant, PEG-PFPE2 (EA surfactant from RAN Biotechnologies), made of poly (ethylene glycol) and perfluoropolyethers (PFPE), is widely used to stabilize the emulsion droplets within fluorinated oil, such that surfactants with a tri-block structure can efficiently form micelles and bilayer vesicles as evidenced by Chowdhury et al.
The shells of the double emulsions can be modified, such that in the case of a water-in-oil-in water double emulsion, solvent extraction removes excess hydrophobic phase from the shell, which can induce other changes in double emulsions such as, for example, their transition into lipid vesicles, polymersomes, or colloidosomes via dewetting or other phenomena (interpreted as micelle formation).
wherein it is known that surfactants can form micelles above the critical micelle concentration, and the formation of micelles is expected in all cases of higher surfactant concentrations in microreactors as evidenced by Schuch et al.
That mixing can partition the sample and bead mixture into a plurality of fluidic droplets (e.g., aqueous droplets within a water-in-oil emulsion), in which at least a subset of the droplets may encapsulate a barcoded bead such as a gel bead; and a microfluidic device that can be used to combine sample with beads (e.g., a set of barcoded beads) as well as an agent capable of degrading the beads (interpreted as delivering a lysis agent to a sample in an aqueous droplet).
The combined references of Delaney and Abate teach all of the limitations of the claims. Thus, the claims remain rejected for the reasons of record.
Regarding (b), please see the discussion supra regarding the Examiner’s response to Applicant’s argument. Applicant’s assertion that the Examiner acknowledges that Delaney fails to teach delivering a micellized lysis agent, is not true. The Examiner has not stated that Delaney fails to teach delivering a micellized lysis agent to the cell. The Examiner has noted that Delaney et al. do not specifically exemplify delivering micellized lysis agent prior to the delivery step. In addition to the teachings cited supra, Delaney and Abate teach:
(1) The controlled delivery of reagents to subsets of sample components including into discrete partitions; that microcapsules are used to deliver additional reagents to a partition; the co-partitioning of a cell and lysis reagents/lysis enzymes (interpreted as delivering a lysis agent to a sample in an aqueous droplet); and micelles comprising lysis agent, wherein surface mediated coalescence in the presence of DBDM can occur (interpreted as delivering a micellized lysis agent to a sample in an aqueous droplet) (Delaney), where it is known that carrier phases with surfactant can undergo micellar transport allowing compounds to be transported into or out of droplets as evidenced by Abate ‘507 (Delaney).
(2) That mixing can partition the sample and bead mixture into a plurality of fluidic droplets (e.g., aqueous droplets within a water-in-oil emulsion), in which at least a subset of the droplets can encapsulate a barcoded bead such as a gel bead (interpreted as partitioning); contacting lysis agents with the cell suspension concurrently with, or immediately prior to the introduction of cells into the partitioning junction/droplet generation zone and/or when the cells have been suspended in the aqueous fluid, or pre-encapsulated, prior to the partitioning process (interpreted as delivering lysis agents to the aqueous droplet); and a microfluidic device that can be used to combine sample with beads such as a set of barcoded beads, as well as an agent capable of degrading the beads (interpreted as delivering a lysis agent to a sample in an aqueous droplet) (Abate).
The combined references of Delaney and Abate teach all of the limitations of the claims. Thus, the claims remain rejected for the reasons of record.
New Objections/Rejections
Claim Objections
Claims 51-53 are objected to because of the following informalities: Claims 51-53 recites the abbreviations such as Sarkosyl, Capstone FS-61, CTAB, Triton X-100, Triton X-114, NP-40, Tween-80, Brij 35, CHAPS, CHAPSO, ASB-14, ASB-16, SB-3-10, and SB-3-12 where an abbreviation should be spelled out in the first encounter of the claims.
Appropriate correction is required.
Conclusion
Claims 31-33, 35, 36, 38-40, 45, 47-49 and 51-56 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMY M BUNKER whose telephone number is (313) 446-4833. The examiner can normally
be reached on Monday-Friday (6am-2:30pm).
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/interviewpracti