DETAILED ACTION
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 .
Status of the Claims
Claims 1-2, 5-7, 10, 31-39, and 41-50 are pending. Claims 1-2, 5-7, 10, and 41-48 are the subject of this Non-Final Office Action. Claims 31-39 and 49-50 have been withdrawn. Claims 45-50 are new.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/21/2026 has been entered.
Election/Restrictions
Claims 49 and 50 have been withdrawn as they are dependent upon withdrawn claim 31.
Response to Arguments
Applicant’s amendments to the claims have overcome the double patenting rejection previously set forth in the Final Office Action of 10/21/2025.
Applicant’s arguments with respect to claim(s) 1, 2, 5,6, 10, 43, and 44 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. In particular, George et al. (US 2014/0079923 A1) teaches the inclusion of N-(5-azioacetamidylpentyl) acrylamide in the beads of Belhocine and Belhocine ‘226, as described below.
New Grounds - Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim(s) 1-2, 5-6, 10, 44-45, and 47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine et al. (US 2018/0216162 A1; filed 02/02/2018 with priority to 1/30/2017; previously cited), Belhocine et al. (WO 2018/218226 A1, filed 05/25/2018 with priority to 05/26/2017; referred to as Belhocine ‘226), and George et al. (US 2014/0079923 A1).
Regarding claims 1 and 44, Belhocine teaches a method for analyzing a single biological particle, wherein the single biological particle is enclosed within a polymer or gel matrix (par. 0013). Belhocine teaches the polymer or gel matrix is diffusively permeable to reagents while retaining the one or more macromolecular constituents (par. 0014, 0132). Belhocine teaches the polymer or gel matrix comprises PEG-acrylate/thiol (par. 0017).
Belhocine teaches that the bead may comprise primers (par. 0056, 0168, 0203), and teaches that the bead may comprise an acrydite moiety which can be attached to an oligonucleotide, such as a primer for amplifying target nucleic acids, to be incorporated into the bead (par. 0157, 210). Belhocine teaches the nucleic acids derived from the cells may include DNA, RNA, or cDNA (par. 0124, 0203, 0215).
Belhocine further teaches that the cell beads can be contacted with lysis reagents to release the contents of cells or viruses, and teaches that the pore size is sufficiently small to retain nucleic acid fragments of a particular size (par. 0201). Belhocine further teaches that other reagents can be contacted with the cell beads, including those useful in modification of a cell bead’s nucleic acid, amplification of a cell bead’s nucleic acid, and attachment of barcodes to the amplified fragments (par. 0203). Belhocine teaches these processes can occur following partitioning of the cell beads into discrete partitions and prior to release of the contents of the cell beads into their respective partitions (par. 0204). This is interpreted as encompassing a library being prepared within the bead matrix.
Belhocine does not state that a tagmentation reaction is performed within the cell beads, and does not teach the inclusion of N-(5-azidoacetamidylpentyl) acrylamide. Belhocine does teach one or more reactions comprising nucleic acid insertion, nucleic acid cleavage, or any combination thereof, and teaches the insertion can comprise transposon-mediated insertion and the cleavage can comprise transposon-mediated cleavage (par. 0051).
Belhocine ‘226 teaches methods and systems for sample preparation techniques that allow amplification of single cells, wherein the preparation of barcoded next-generation sequencing libraries is facilitated by transposon-mediated transposition and fragmentation of a target nucleic acid sequence (Abstract).
Belhocine ‘226 teaches that species may be encapsulated in beads during bead generation, and includes reagents for template preparation such as tagmentation (par. 00175).
It would have been obvious to one of ordinary skill in the art to substitute the transposon-mediated insertion and cleavage as taught by Belhocine for the tagmentation as taught by Belhocine ‘226, as Belhocine ‘226 teaches that these reagents can be encapsulated in beads and would be a simple substitution for the transposon-mediated insertion and transposon-mediated cleavage as taught by Belhocine, with no evidence of unexpected results.
Belhocine teaches that beads may be functionalized for attachment of oligonucleotides (par. 0156-0158).
George teaches a method of preparing a substrate surface by using beads coated with a covalently attached polymer such as poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide), referred to as PAZAM (Abstract, par. 0363, 0376). George teaches that substrates having PAZAM-coated surfaces are used to determine a nucleotide sequence of a polynucleotide (par. 0036). George further teaches that molecular analyses, such as certain nucleic acid sequencing methods rely on the attachment of nucleic acid strands to a polymer-coated surface of a substrate (par. 0003).
It would have been obvious to one of ordinary skill in the art to include N-(5-azioacetamidylpentyl) acrylamide in the hydrogel beads of Belhocine. Belhocine teaches that the beads may be functionalized for the attachment of oligonucleotides (par. 0156), including activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production (par. 0156). George teaches that N-(5-azioacetamidylpentyl) acrylamide is used for the attachment of nucleic acid strands to a polymer-coated surface and teaches that it allows for molecular analyses such as certain nucleic acid sequencing method. Therefore, it would have been obvious to one of ordinary skill in the art to substitute one method for attachment of oligonucleotides for another, along with the benefit of allowing for certain molecular analyses as taught by George, with no evidence of unexpected results.
Claim 2 recites the limitation that “the bead has a diameter of about 2 µm to about 120 µm.” Belhocine teaches that the diameter of a bead can be about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm, and teaches that in some cases, a bead may have a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm. These values encompass the claimed diameter; it would therefore be obvious to one of ordinary skill in the art to select a diameter of about 2 µm to about 120 µm, as Belhocine teaches these diameters of beads.
Regarding claims 5 and 6, Belhocine discloses the cells may include bacterial or mammalian cells (par. 0116).
Regarding claim 10, Belhocine teaches the cell bead comprises reagents such as nucleotides (par. 0056), and gives the example of nucleotides or exogenous chemicals such as NaOH as reagents (par. 0056, 0330). Belhocine further discloses reagents may be chemicals, primers, or enzymes including primers, transposons, and proteinase K (par. 0056, 0202).
Regarding claims 45 and 47, Belhocine teaches that the bead may comprise primers (par. 0056, 0168, 0203), and teaches that the bead may comprise an acrydite moiety which can be attached to an oligonucleotide, such as a primer for amplifying target nucleic acids, to be incorporated into the bead (par. 0157, 210).
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine, Belhocine ‘226, and George as applied to claim 5 above, and further in view of Andersen et al. (US 2012/0208255 A1; previously cited).
Regarding claim 7, Belhocine does not teach that the cell is an Escherichia coli cell, a Bacillus subtilis cell, an Aeromonas hydrophila cell, or a fibroblast cell. Belhocine teaches the cell may be any type of cell (par. 0116).
Andersen teaches a cell encapsulation mixture formed into beads with pores (par. 0056). Andersen discloses that two or more materials may be used as the encapsulation medium, such as copolymers of poly(ethylene oxide) and polypropylene oxide (par. 0040). Andersen further discloses that the resulting polymerized matrix contains pores that are of sufficient size to retain cells but allow the unrestricted movement of sugar into the bead and ethanol out (par. 0060).
Andersen teaches the cells may be bacterial cells including Escherichia coli (par. 0068). It would have been obvious to one of ordinary skill in the art to substitute to the bacterial cell of Belhocine for the E. coli cell of Andersen, as this would be a simple substitution of one bacterial cell for another, with no evidence of unexpected results.
Claim(s) 41 and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine, Belhocine '226, and George as applied to claim 1 above, and further in view of Hao et al. (Mechanical behavior of hybrid hydrogels composed of a physical and a chemical network. Polymer. 54, 2013, 2174-2182; previously cited).
Belhocine, Belhocine ‘226, and George do not teach the inclusion of isopropyl alcohol in the hydrogel polymer.
Hao teaches that hydrogels are composed of chemically and/or physically-crosslinked hydrophilic polymers, and that most conventional hydrogels employ a single chemical or physical network. Hao further teaches that chemical networks formed from covalent bonds have permanent shapes but poor mechanical strength (pg. 2174, left col., par. 1). Hao teaches that hybrid hydrogels had higher stiffness and strength but lower extensibility compared to a physical hydrogel without covalent crosslinks (Abstract).
Hao teaches the hybrid hydrogels were prepared in 15 wt% isopropanol solutions (pg. 2175, right col., par. 2). Hao teaches the use of isopropanol allows for the covalent network to be developed first (pg. 2177, right col., par. 2).
It would have been obvious to one of ordinary skill in the art to include isopropyl alcohol in the hydrogel polymer, as Hao teaches the inclusion of isopropyl alcohol in the formation of hydrogels allows for the formation of a hybrid hydrogel with both physical and covalent crosslinks (pg. 2175, right col., par. 2; Abstract).
One of ordinary skill in the art would have been motivated to include isopropyl alcohol to form a hybrid hydrogel as Hao teaches that some properties of a hybrid hydrogel, such as recovery from deformation, are superior to conventional hydrogels (Abstract; pg. 2182, left col., par. 2-3; pg. 2182, right col., par. 1-2).
Regarding claim 42, Hao teaches the use of 15 wt% isopropanol solutions (pg. 2175, right col., par. 2). Additionally, it would have been obvious to one of ordinary skill in the art to optimize the amount of isopropyl alcohol in order to achieve a hydrogel with the desired properties, with no evidence of unexpected results.
Claim(s) 43 is/are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine, Belhocine ‘226, and George as applied to claim 1 above, and further in view of Klann et al. (US 2009/0130756 A1; previously cited).
Regarding claim 43, Belhocine does not teach a porosity between about 0.8 to about 0.95.
Belhocine teaches that the cell beads have a tunable pore size which may be chosen to retain denatured nucleic acids but maintain diffusive permeability to exogenous chemicals such as NaOH and/or endogenous chemicals such as inhibitors (par. 0330).
Klann teaches the formation of hydrogel matrices for the cryopreservation of cells (Abstract). Klann teaches that hydrogels comprise polyethylene glycol (par. 0009, 0091). Klann teaches that the porosity of crosslinked hydrogels can be controlled by a variety of methods (par. 0105). Klann further teaches that the average for size of the hydrogel matrix particles can vary depending upon the desired use, and the porosity can vary as well (par. 0128-0129, 0150). Klann teaches that the porosity can be about 30% to about 90%, about 40% to about 85%, or about 50% to about 80% (par. 0112).
It would have been obvious to one of ordinary skill in the art to vary the porosity of the hydrogel beads as taught by Belhocine to achieve a porosity of about 0.8 to about 0.95, as Klann teaches that the porosity of hydrogel matrices can be varied depending upon the desired use, and provides ranges of porosity which overlap with the claimed range. Additionally, it would be obvious to one of ordinary skill in the art to vary the porosity in order to optimize the hydrogel beads for its desired use, with no evidence of unexpected results.
Claim(s) 46 and 48 is/are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine, Belhocine ‘226, and George as applied to claims 45 and 47 above, and further in view of Breed et al. (Functionalization of polymer microspheres using click chemistry. Langmuir. 2009, 25(8), 4370-4376).
Regarding claims 46 and 48, George teaches that primers can be attached to the polymer (par. 0010), and teaches that beads coated with PAZAM can be grafted to primers via reacting the azide groups on PAZAM with a 5’ alkyne modified oligonucleotide (par. 0364 ). George further teaches the use of a copper catalyst (par. 0257).
George does not teach that the primers are chemically linked to the bead using copper-catalyzed azide-alkyne cycloaddition chemistry.
Breed teaches a method of functionalizing microspheres with azides, which serve as an effective handle that can be specifically functionalized by the copper-catalyzed azide-alkyne cycloaddition reaction, which is known for its robustness and compatibility with a range of reaction conditions (pg. 4371, left col., par. 2).
It would have been obvious to one of ordinary skill in the art to use a copper-catalyzed azide-alkyne cycloaddition reaction to chemically link the primers to the beads, as George teaches an azide-alkyne reaction, and Breed teaches that the copper-catalyzed azide-alkyne cycloaddition reaction is well-known and has the benefits of being robust and compatible with a range of reaction conditions, with no evidence of unexpected results.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Randi L Beil whose telephone number is (571)272-1147. The examiner can normally be reached M-F 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, Heather Calamita can be reached at 571-272-2876. 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.
/R.L.B./Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
Prosecution Insights