Granted Patent
B2
US 12,480,090 · App. 17/663,485 · Granted Nov 25, 2025
Contiguity particle formation and methods of use
Inventors:
Frank J. Steemers (Encinitas, CA); Ramesh Ramji (San Diego, CA); Steven Norberg (La Mesa, CA); Lena Christiansen (San Diego, CA); Dmitry K. Pokholok (San Diego, CA); Fan Zhang (San Diego, CA)
Assignee:
ILLUMINA, INC.
View Patent ↗
Loading inventors, assignments & file history…
Abstract
Embodiments of systems, methods, and compositions provided herein relate to hollow beads encapsulating single cells. Some embodiments include performing multiple co-assays on a single cell encapsulated within a hollow bead, including nucleic acid sequencing, preparing nucleic acid libraries, determining methylation status, identifying genomic variants, or protein analysis.
Claims (25)
1 . A method of encapsulating a single cell within a hollow bead, comprising:
combining a single cell with a polymer in a spacer oil to form a mixture (a);
mixing the mixture (a) with a crosslinking oil to form a porous polymer shell encapsulating the single cell; and
modulating the pore size of the polymer shell, thereby allowing diffusion of reagents through the polymer shell while retaining the single cell,
wherein the mixing comprises inputting the single cell, the polymer, the spacer oil, and the crosslinking oil into a droplet generator, and
wherein the reagents comprise lysing agents, random hexamers, polymerases, transposases, ligases, catalyzing enzymes, deoxynucleotide triphosphates, buffers, divalent cations, primers, or a combination thereof.
2 . The method of claim 1 , wherein the hollow bead has a diameter of about 20 μm to about 200 μm.
3 . The method of claim 1 , wherein the polymer comprises a four-arm polyethylene glycol (PEG).
4 . The method of claim 3 , wherein the four-arm polyethylene glycol (PEG) is selected from the group consisting of PEG-acrylate, PEG-amine, PEG-carboxylate, PEG-dithiol, PEG-epoxide, PEG-isocyanate, and PEG-maleimide.
5 . The method of claim 1 , wherein the spacer oil comprises mineral oil or fluorocarbon oil.
6 . The method of claim 1 , wherein the crosslinking oil comprises dithiol or amine dissolved in oil.
7 . The method of claim 1 , wherein the single cell is a mammalian cell.
8 . The method of claim 1 , wherein the primers have a size of less than 50 base pairs.
9 . The method of claim 1 , wherein the droplet generator is a microfluidic chip.
10 . The method of claim 1 , wherein the single cell is fixed prior to mixing by contact with a fixative.
11 . The method of claim 10 , wherein the fixative comprises an alcohol or an aldehyde.
12 . The method of claim 11 , wherein the alcohol is methanol or ethanol.
13 . The method of claim 11 , wherein the aldehyde is paraformaldehyde.
14 . The method of claim 1 , wherein an interior of the polymer shell comprises an aqueous environment.
15 . The method of claim 1 , wherein the single cell encapsulated within the polymer shell is free from interaction with the polymer shell and/or is not in contact with the polymer shell.
16 . The method of claim 1 , wherein the hollow bead encapsulating the single cell is seeded on a solid support.
17 . The method of claim 16 , wherein the solid support is an etched surface, a well, a flow-cell device, a microfluidic channel, a bead, or a column.
18 . The method of claim 1 , further comprising permeabilizing the encapsulated cell with a detergent.
19 . The method of claim 1 , wherein the modulating the pore size of the polymer shell comprises changing salt concentration, pH, or temperature.
20 . The method of claim 1 , wherein the lysing agents comprise lysozyme, glucolase, zymolase, lyticase, proteinase K, proteinase E, a viral endolysin, or an exolysin.
Assignments (2)
CORRECTIVE ASSIGNMENT TO CORRECT THE DOC DATE FOR ASSIGNOR FAN ZHANG FROM 08/24/2019 TO 08/23/2019 PREVIOUSLY RECORDED AT REEL: 059934 FRAME: 0407. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT.
Recorded May 23, 2022
From: STEEMERS, FRANK J.; RAMJI, RAMESH; NORBERG, STEVEN; CHRISTIANSEN, LENA; POKHOLOK, DMITRY K.; ZHANG, FAN
To: ILLUMINA, INC.
Reel/Frame 060161/0305 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 17, 2022
From: STEEMERS, FRANK J.; RAMJI, RAMESH; NORBERG, STEVEN; CHRISTIANSEN, LENA; POKHOLOK, DMITRY K.; ZHANG, FAN
To: ILLUMINA, INC.
Reel/Frame 059934/0407 →
Continuity (3)
Provisional Application
62660452
· Apr 20, 2018
References Cited (279)
US 3957741A
· Rembaum et al.
· 1976
[cited by applicant]
US 3985632A
· Rembaum et al.
· 1976
[cited by applicant]
US 4046720A
· Rembaum et al.
· 1977
[cited by applicant]
US 5130238A
· Malek
· 1992
[cited by applicant]
US 5185243A
· Ullman
· 1993
[cited by applicant]
US 5455166A
· Walker
· 1995
[cited by applicant]
US 5573907A
· Carrino
· 1996
[cited by applicant]
US 5679524A
· Nikiforov
· 1997
[cited by applicant]
US 5958451A
· Chen
· 1999
[cited by applicant]
US 6210891B1
· Nyren
· 2001
[cited by applicant]
US 6214587B1
· Dattagupta
· 2001
[cited by applicant]
US 6258568B1
· Nyren
· 2001
[cited by applicant]
US 6274320B1
· Rothberg
· 2001
[cited by applicant]
US 6355431B1
· Chee
· 2002
[cited by applicant]
US 6890741B2
· Fan
· 2005
[cited by applicant]
US 6913884B2
· Stuelpnagel
· 2005
[cited by applicant]
US 7001792B2
· Sauer
· 2006
[cited by applicant]
US 7057026B2
· Barnes
· 2006
[cited by applicant]
US 7115400B1
· Adessi
· 2006
[cited by applicant]
US 7211414B2
· Hardin
· 2007
[cited by applicant]
US 7244559B2
· Rothberg
· 2007
[cited by applicant]
US 7315019B2
· Turner
· 2008
[cited by applicant]
US 7329492B2
· Hardin
· 2008
[cited by applicant]
US 7405281B2
· Xu
· 2008
[cited by applicant]
US 7582420B2
· Oliphant
· 2009
[cited by applicant]
US 7595883B1
· Gamal
· 2009
[cited by applicant]
US 7611869B2
· Fan
· 2009
[cited by applicant]
US 7670810B2
· Gunderson
· 2010
[cited by applicant]
US 7985565B2
· Mayer
· 2011
[cited by applicant]
US 8003354B2
· Shen et al.
· 2011
[cited by applicant]
US 9096899B2
· Helmy et al.
· 2015
[cited by applicant]
US 9388465B2
· Hindson et al.
· 2016
[cited by applicant]
US 9694361B2
· Bharadwaj et al.
· 2017
[cited by applicant]
US 9951386B2
· Hindson et al.
· 2018
[cited by applicant]
US 20030175824A1
· Pishko et al.
· 2003
[cited by applicant]
US 20050053980A1
· Gunderson
· 2005
[cited by applicant]
US 20050181440A1
· Chee
· 2005
[cited by applicant]
US 20050191698A1
· Chee
· 2005
[cited by applicant]
US 20090061009A1
· Schwarz et al.
· 2009
[cited by applicant]
US 20090130756A1
· Klann
· 2009
[cited by applicant]
US 20100137143A1
· Rothberg
· 2010
[cited by applicant]
US 20120129954A1
· Falcone et al.
· 2012
[cited by applicant]
US 20120208255A1
· Andersen et al.
· 2012
[cited by applicant]
US 20130011441A1
· Hollinger et al.
· 2013
[cited by applicant]
US 20140378349A1
· Hindson et al.
· 2014
[cited by applicant]
US 20150284768A1
· Craig et al.
· 2015
[cited by applicant]
US 20150376609A1
· Hindson et al.
· 2015
[cited by applicant]
US 20220106588A1
· Wu et al.
· 2022
[cited by applicant]
US 20220411859A1
· Wu et al.
· 2022
[cited by applicant]
CA 2622955
· 2007
[cited by applicant]
CA 3047328
· 2018
[cited by applicant]
CN 101514956
· 2009
[cited by applicant]
CN 101838696
· 2010
[cited by applicant]
CN 101845494
· 2010
[cited by applicant]
CN 102102122
· 2011
[cited by applicant]
CN 103328981
· 2013
[cited by applicant]
CN 104471075
· 2015
[cited by applicant]
CN 105412935
· 2016
[cited by applicant]
CN 105431554
· 2016
[cited by applicant]
CN 105492607
· 2016
[cited by applicant]
CN 105916689
· 2016
[cited by applicant]
CN 106536734
· 2017
[cited by applicant]
CN 106769336
· 2017
[cited by applicant]
CN 107002117
· 2017
[cited by applicant]
CN 107281497
· 2017
[cited by applicant]
CN 108165119
· 2018
[cited by applicant]
EP 0320308
· 1989
[cited by applicant]
EP 0336731
· 1989
[cited by applicant]
EP 0439182
· 1991
[cited by applicant]
JP 2016519079
· 2007
[cited by applicant]
JP 2007112754
· 2007
[cited by applicant]
JP 4699207B2
· 2011
[cited by applicant]
JP 2016517281
· 2016
[cited by applicant]
JP 2017510292
· 2017
[cited by applicant]
JP 2020532948
· 2020
[cited by applicant]
KR 1020130022671
· 2013
[cited by applicant]
KR 1020170020704
· 2017
[cited by applicant]
RU 2274863C2
· 2006
[cited by applicant]
RU 2603745
· 2016
[cited by applicant]
RU 2736728
· 2020
[cited by applicant]
WO WO198909835
· 1989
[cited by applicant]
WO WO198912696
· 1989
[cited by applicant]
WO WO199001069
· 1990
[cited by applicant]
WO WO1991006678
· 1991
[cited by applicant]
WO WO9523875
· 1995
[cited by applicant]
WO WO200002018
· 2000
[cited by applicant]
WO WO01051196
· 2001
[cited by applicant]
WO WO02017888
· 2002
[cited by applicant]
WO WO03078659
· 2003
[cited by applicant]
WO WO03079401
· 2003
[cited by applicant]
WO WO2004018497
· 2004
[cited by applicant]
WO WO05021755
· 2005
[cited by applicant]
WO WO05021804
· 2005
[cited by applicant]
WO WO05032512
· 2005
[cited by applicant]
WO WO06062609
· 2006
[cited by applicant]
WO WO2006125458
· 2006
[cited by applicant]
WO WO07025124
· 2007
[cited by applicant]
WO WO07114758
· 2007
[cited by applicant]
WO WO2007123744
· 2007
[cited by applicant]
WO WO08045806
· 2008
[cited by applicant]
WO WO2008109176
· 2008
[cited by applicant]
WO WO10003132
· 2010
[cited by applicant]
WO WO11123246
· 2011
[cited by applicant]
WO WO11156432
· 2011
[cited by applicant]
WO WO2012058096
· 2012
[cited by applicant]
WO WO13036847
· 2013
[cited by applicant]
WO WO14152673
· 2014
[cited by applicant]
WO WO2014145555
· 2014
[cited by applicant]
WO WO2014189957
· 2014
[cited by applicant]
WO WO2015048173
· 2015
[cited by applicant]
WO WO2015088299
· 2015
[cited by applicant]
WO WO15121434
· 2015
[cited by applicant]
WO WO14133905
· 2015
[cited by applicant]
WO WO2015147147
· 2015
[cited by applicant]
WO WO2015200893
· 2015
[cited by applicant]
WO WO16004068
· 2016
[cited by applicant]
WO WO2016004234
· 2016
[cited by applicant]
WO WO2016130704
· 2016
[cited by applicant]
WO WO16160475
· 2016
[cited by applicant]
WO WO2017013138
· 2017
[cited by applicant]
WO WO2017040024
· 2017
[cited by applicant]
WO WO17075072
· 2017
[cited by applicant]
WO WO2017075265
· 2017
[cited by applicant]
WO WO2017100347
· 2017
[cited by applicant]
WO WO18052464
· 2018
[cited by applicant]
WO WO2018071448
· 2018
[cited by applicant]
WO WO18118971
· 2018
[cited by applicant]
WO WO2018119301
· 2018
[cited by applicant]
WO WO18156519
· 2018
[cited by applicant]
WO WO2018140966
· 2018
[cited by applicant]
WO WO18172726
· 2018
[cited by applicant]
WO WO18200867
· 2018
[cited by applicant]
WO WO18203141
· 2018
[cited by applicant]
WO WO2019028047
· 2019
[cited by applicant]
WO WO2019028166
· 2019
[cited by applicant]
WO WO2019160820
· 2019
[cited by applicant]
WO WO20112604
· 2020
[cited by applicant]
WO WO20165433
· 2020
[cited by applicant]
WO WO20167866
· 2020
[cited by applicant]
Hodne, K et al. Single-cell isolation and gene analysis: Pitfalls and possibilities. Int. J. Mol. Sci. 2015. 16: 26832-26849. (Year: 2015).
[cited by examiner]
Yu, J et al. Synthesis and characterization of MMP degradable and maleimide cross-linked PEG hydrogels for tissue engineering scaffolds. Polymer Degradation and Stability. 2016. 133: 312-320. (Year: 2016).
[cited by examiner]
Ramli, Ros Azlinawati,et al., “Core-shell polymers: a review”, RSC Adv., 2013, 3, 15543-15565.
[cited by applicant]
Notice of Allowance issued in RU application No. 2021118349, dated Feb. 3, 2023.
[cited by applicant]
Office Action issued in RU application No. 2021118349, dated Sep. 14, 2022.
[cited by applicant]
Office Action issued in JP application No. 2022-021757, dated Mar. 7, 2023.
[cited by applicant]
Appleby et al. (Methods Mol Biol. 2009; 513:19-39).
[cited by applicant]
Augst et al. Alginate hydrogels as biomaterials. Macromolecular Bioscience. 2006. 6: 623-633. (Year: 2006).
[cited by applicant]
Bentley et al., Nature 456:53-59 (2008).
[cited by applicant]
Bigdeli et al. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification. PLoS One. 2015. 10(2): e011738. 15 pages. (Year: 2015).
[cited by applicant]
Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008).
[cited by applicant]
Deamer et al. Acc. Chem. Res. 35:817-825 (2002).
[cited by applicant]
Deamer et al. Trends Biotechnol. 18, 147-151 (2000).
[cited by applicant]
Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002).
[cited by applicant]
Decision to Grant issued in RU application No. 2019144343, dated Apr. 27, 2021.
[cited by applicant]
Elbert, DL et al. Protein delivery from materials formed by self-selective conjugate additions reactions. Journal of Controlled Release. 2001. 76: 11-25. (Year: 2001).
[cited by applicant]
Fox et al. (Methods Mol Biol. 2009;553:79-108).
[cited by applicant]
Freeman et al: “Single-cell genome sequencing at ultra-high-throughput with microfluidic dropletbarcoding. (includes Online Methods)”, 1-7, 24-33, Nature Biotechnology, vol. 35, No. 7, Jul. 1, 2017 (Jul. 1, 2017) pp. 64…
[cited by applicant]
Garni, Biopores/membrane proteins in synthetic polymer membranes, Biochimica et Biophysica Acta, 1859, 2017, 619-638.
[cited by applicant]
Grothues et al. Nucleic Acids Res. 21(5):1321-2 (1993).
[cited by applicant]
Healy, Nanomed. 2, 459-481 (2007).
[cited by applicant]
International Search Report and Written Opinion issued in patent application No. PCT/US2019/027540, mailed Jul. 24, 2019.
[cited by applicant]
International Search Report issued in application No. PCT/US2018/044855, dated Oct. 15, 2018.
[cited by applicant]
Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008).
[cited by applicant]
Kumachev et al., “High-throughput generation of hydrogel microbeads with varying elasticity for cell encapsulation”, Biomaterials, Elsevier Science Publishers BV., Barking, GB, (Feb. 1, 2011), pp. 1477-1483.
[cited by applicant]
Lage et al., Genome Research 13:294-307 (2003).
[cited by applicant]
Levene et al. Science 299, 682-686 (2003).
[cited by applicant]
Li et al. Nat. Mater. 2:611-615 (2003).
[cited by applicant]
Lizardi et al., Nat. Genet. 19:225-232 (1998).
[cited by applicant]
Lundquist et al. Opt. Lett. 33, 1026-1028 (2008).
[cited by applicant]
Margulies et al. (Nature 2005 437: 376-80).
[cited by applicant]
Morozova et al. (Genomics. 2008 92:255-64).
[cited by applicant]
Novak et al., “Single-cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions”, WILEY-VCH Verlag GMBH & co, DE, vol. 50, No. 2, Jan. 10, 2011 (Jan. 10, 2011), pp. 390-395.
[cited by applicant]
Office Action issued in JP application No. 2019-568626, dated Feb. 2, 2021.
[cited by applicant]
Picelli et al., “Tn5 transposase and tagmentation procedures for massively scaled sequencing projects”, Genome Research, vol. 24, No. 12, Jul. 30, 2014 (Jul. 30, 2014), pp. 2033-2040.
[cited by applicant]
Pregibon et al., “Optimization of Encoded Hydrogel Particles for Nucleic Acid Quantification”, Analytical Chemistry, vol. 81, No. 12, Jun. 15, 2009 (Jun. 15, 2009), pp. 4873-4881.
[cited by applicant]
Rakszewska et al. (Angewandte Chemie, 2016, 55:6698-6701) (Year: 2016).
[cited by applicant]
Ronaghi et al. (Analytical Biochemistry 1996 242: 84-9).
[cited by applicant]
Ronaghi et al. Science 281(5375), 363 (1998).
[cited by applicant]
Ronaghi, Genome Res. 11(1), 3-11 (2001).
[cited by applicant]
Search Report and Written Opinion issued in application No. PCT/US2019/057852, mailed Jan. 16, 2020.
[cited by applicant]
Search Report issued in RU application No. 2019144343, dated Jun. 21, 2020.
[cited by applicant]
Search Report issued in AU application No. 2019220559, dated Mar. 12, 2021.
[cited by applicant]
Search Report issued in AU application No. 2019220559, dated May 19, 2021.
[cited by applicant]
Search Report issued in AU application No. 2018312560, dated Mar. 11, 2021.
[cited by applicant]
Office Action issued in EP application No. 18755643.6, dated May 10, 2021.
[cited by applicant]
Office Action issued in JP application No. 2019-568626, dated Jun. 22, 2021.
[cited by applicant]
Shendure et al. (Science 2005 309: 1728-32).
[cited by applicant]
Shupletsova et al., “Encapsulation of cells and tissues of the pancreas: problems and ways to overcome them”, Genes and cells, vol. XI, N 1, 2016, pp. 18-23.
[cited by applicant]
Soni et al. Clin. Chem. 53, 1996-2001 (2007).
[cited by applicant]
Tan et al , Heterogeneous multi-compartmental hydrogel particles as synthetic cells for incompatible tandem reactions, 2017, Nature Communications, 8,663, pp. 1-10 (Year: 2017).
[cited by applicant]
Trivedi et al. Microfluidic encapsulation of cells in alginate capsules for high throughput screening. 31st Annual International Conference of the IEEE Embs. Minneapolis, Minnesota, USA. Sep. 2-6, 2009. 2009: 7037-7040.…
[cited by applicant]
Vitak et al. Nat Meth. 2017;14:302-308.
[cited by applicant]
Walker et al., Nucl. Acids Res. 20:1691-96 (1992).
[cited by applicant]
Boeke et a., 1989, Transcription and reverse transcription of retrotransposons, Annu Rev Microbiol. 43:403-434.
[cited by applicant]
Brown et al., 1989, Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein, Proc Natl Acad Sci USA, 86:2525-2529.
[cited by applicant]
Colegio et al., 2001, In vitro transposition system for efficient generation of random mutants of campylobacter jejuni, J. Bacteriol., 183(7):2384-2388.
[cited by applicant]
Craig, 1996, Transposon Tn7, Review in: Curr Top Microbiol Immunol., 204:27-48.
[cited by applicant]
Craig, Mar. 15, 1996, V(D)J recombination and transposition: closer than expected Science. 271: 1512.
[cited by applicant]
Devine et al., 1994, Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis, Nucleic Acids Res., 22(18):3765-3772.
[cited by applicant]
Gloor, 2004, Gene targeting in
[cited by applicant]
Goryshin et al., Mar. 27, 1998, Tn5 in vitro transposition, J. Biol. Chem. 273:7367-7374.
[cited by applicant]
Ichikawa et al., Nov. 5, 1990, In vitro transposition of Transposon Tn3*, J. Biol. Chem., 265:18829-18832.
[cited by applicant]
Kirby et al., 2002, Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue, Mol. Microbiol., 43(1):173-186.
[cited by applicant]
Kleckner et al., 1996, Tn10 and IS10 transposition and chromosome rearrangements: mechanism and regulation in vivo and in vitro, Curr. Top. Microbiol. Immunol., 204:49-82.
[cited by applicant]
Lampe et al., 1996, A purified mariner transposase is sufficient to mediate transposition in vitro, EMBO J., 15:5470-5479.
[cited by applicant]
Mizuuchi, Dec. 1983, In vitro transposition of bacteriophage mu: a biochemical approach to a novel replication reaction, Cell, 35:785-794.
[cited by applicant]
Ohtsubo 1996, Bacterial insertion sequences, Curr. Top. Microbiol. Immunol. 204:1-26.
[cited by applicant]
Plasterk, 1996, The Tc1/mariner transposon family, Curr. Top. Microbiol. Immunol., 204:125-143.
[cited by applicant]
Savilahti et al., 1995, The phage mu transposone core: DNA requirements for assembly and function, EMBO J., 14:4893-4903.
[cited by applicant]
Wikipedia, Polyethylene glycol, 8 pages, revision date Jul. 4, 2016 by user “Anypodetos”. [Retrieved on Jul. 25, 2023]. Retrieved from Internet: < URL:https://en.wikipedia.org/w/index.php?title=Polyethylene_glycol&oldid…
[cited by applicant]
Wilson et al., 2007, New transposon delivery plasmids for insertional mutagenesis in bacillus anthracis, J. Microbiol. Methods, 71:332-335.
[cited by applicant]
Zhang et al., Oct. 2009, A novel mechanism of transposon-mediate gene activation, PLoS Genet. 5(10):e1000689.
[cited by applicant]
Examination report dated Jun. 6, 2023 in Indian patent application No. 202117010489.
[cited by applicant]
Office Action dated Apr. 24, 2023 in Russian patent application No. 2021108033/04(017322), 17 pp.
[cited by applicant]
Adachi et al. Nov. 1-5, 2009, Hydrogel microbeads for high throughput PCR, Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences, https://www.rsc.org/binaries/LOC/2009/Pdf/581-W21F.…
[cited by applicant]
Choi et al., Jun. 20, 2007, Generation of monodisperse alginate microbeads and in situ encapsulation of cell in microfluidic device, Biomedical Microdevices, 9(6):855-862.
[cited by applicant]
Head et al., Feb. 2014, Library construction for next-generation sequencing: Overviews and challenges, BioTechniques, 56:61-77.
[cited by applicant]
Heo et al., Dec. 3, 2002, A microfluidic bioreactor based on hydrogel-entrapped
[cited by applicant]
Li et al., Jan. 26, 2015, Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy, Journal of the American Chemical Society, 137(4):1412-1415.
[cited by applicant]
Mitra et al., Sep. 2003, Fluorescent in situ sequencing on polymerase colonies, Analytical Biochemistry, 320(1):55-65.
[cited by applicant]
Quail et al., Jul. 24, 2012, A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers, BioMed Central, 13.
[cited by applicant]
Rakszewski et al., Oct. 3, 2014, One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis, NPG Asia Materials, 6:e133, 11 pp.
[cited by applicant]
Steinhilber et al., Nov. 29, 2013, A microgel construction kit for biorthogonal encapsulation and pH-controlled release of living cells, Angewandte Chemie International Edition, 52(51):13538-13543.
[cited by applicant]
Walser et al., Mar. 12, 2009, Novel method for high-throughput colony PCR screening in Nanoliter-reactors, Nucleic Acids Research, 37(8):E57, 8 pp.
[cited by applicant]
Zhu et al., Dec. 28, 2016, Hydrogel droplet microfluidics for high-throughput single molecule/cell analysis, Accounts of Chemical Research, 50:22-31.
[cited by applicant]
Extended European search report dated Sep. 25, 2023 in application No. 23187175.7.
[cited by applicant]
Examination report dated May 17, 2023 in European patent application No. 22178045.5.
[cited by applicant]
Notice to submit response dated Sep. 13, 2024 in Korean patent application No. 10-2021-7009646.
[cited by applicant]
Hao et al., 2013, Mechanical behavior of hybrid hydrogels composed of a physical and a chemical network, Polymer, 54:2174-2182.
[cited by applicant]
Wang et al., 2014, A Versatile Method for Encapsulating Large-Sized DNA into Small-Sized Bioreducible Nanocapsules, J. Phys. Chem. B, 18:3893-3989.
[cited by applicant]
Yom-Tov et al., 2016, PEG-thiol based hdyrogels with controllable properties, European Polymer Journal, 74:1-12.
[cited by applicant]
Notice of reasons for refusal dated May 28, 2024 in Japanese patent application No. 2023-061984.
[cited by applicant]
International search report and written opinion dated May 9, 2022 in application No. PCT/US2022/015113.
[cited by applicant]
Office action dated Mar. 22, 2024 in Chinese patent application No. 201980003334.5.
[cited by applicant]
Office Action dated Sep. 25, 2023 in Russian patent application No. 2021108033/04(017322), 13 PP.
[cited by applicant]
Notice of reasons for rejection dated Sep. 6, 2023 in Japanese patent application No. 2021- 515482.
[cited by applicant]
Notice of reasons for rejection dated Jan. 31, 2024 in Japanese patent application No. 2021-515482.
[cited by applicant]
Patent examination report 1 dated Oct. 13, 2023 in New Zealand patent application No. 774263, 7 pp.
[cited by applicant]
Avens et al., Oct. 17, 2008, Polymerization behavior and polymer properties of eosin-mediated surface modification reactions, Polymer (Guildf), 49(22):4762-4768.
[cited by applicant]
Brandl et al., 2007, Rational design of hydrogels for tissue engineering: impact of physical factors on cell behavior. Biomaterials. 28:134-146.
[cited by applicant]
Chen et al. Sep. 10, 2012, Low modulus biomimetic microgel particles with high loading of hemoglobin. Biomacromolecules, 13(9):2748-2759.
[cited by applicant]
Christiansen et al., 2017, Contiguity-Preserving Transposition Sequencing (CPT-Seq) for Genome-Wide Haplotyping, Assembly, and Single-Cell ATAC-Seq, Methods Mol Biol., 1551:207-221.
[cited by applicant]
Cruise et al., 1998, Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels, Biomaterials, 19:1287-1294.
[cited by applicant]
Garafutdinov et al., 2012, Solid-phase polymerase chain reaction, Bulletin of the Bashkir University, 12(4): 1745-1748.
[cited by applicant]
Imelfort et al., 2009, De novo sequencing of plant genomes using second-generation technologies, Brief Bioinform, 10(6):609-618.
[cited by applicant]
Ioannidis, 2009, Manufacturing of agarose-based chromatographic adsorbents with controlled pore and particle size, a thesis submitted to The University of Birmingham for the degree of Doctor of Philosophy, 214 pp.
[cited by applicant]
Kapur et al., 1996, Hydrodynamic permeability of hydrogels stabilized within porous membranes. Industrial & Engineering Chemistry Research, 35:3179-3185.
[cited by applicant]
Seidlits et al., 2010, The effects of hyaluronic acid hydrogels with tunable mechanical properties on neural progenitor cell differentiation, Biomaterials, 31:3930-3940.
[cited by applicant]
Vivancos et al., 2010, Strand-specific deep sequencing of the transcriptome, Genome Research, 20:989-999.
[cited by applicant]
Walker et al., 1995, A chemiluminescent DNA probe test based on strand displacement amplification, in Wsiedbrauk et al., eds., Molecular Methods for Virus Detection, Academic Press, Inc., p. 330 349.
[cited by applicant]
Weber et al., Sep. 1, 2009. Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function, Journal of Biomedical Materials Research Part A. 90:720-729.
[cited by applicant]
Office Action issued in JP application No. 2023-19838, dated Apr. 22, 2025, 4 pp.
[cited by applicant]
Official Action dated Jun. 27, 2023 in Japanese patent application No. 2022-063500, 4 pp.
[cited by applicant]
Official action of the substantive examination dated Jun. 10, 2025 in Russian patent application No. 2023119498/10(042053).
[cited by applicant]