title: Analysis of single nucleic acid molecules in micro- and nano-fluidics.
abstract: Nucleic acid analysis has enhanced our understanding of biological processes and disease progression, elucidated the association of genetic variants and disease, and led to the design and implementation of new treatment strategies. These diverse applications require analysis of a variety of characteristics of nucleic acid molecules: size or length, detection or quantification of specific sequences, mapping of the general sequence structure, full sequence identification, analysis of epigenetic modifications, and observation of interactions between nucleic acids and other biomolecules. Strategies that can detect rare or transient species, characterize population distributions, and analyze small sample volumes enable the collection of richer data from biosamples. Platforms that integrate micro- and nano-fluidic operations with high sensitivity single molecule detection facilitate manipulation and detection of individual nucleic acid molecules. In this review, we will highlight important milestones and recent advances in single molecule nucleic acid analysis in micro- and nano-fluidic platforms. We focus on assessment modalities for single nucleic acid molecules and highlight the role of micro- and nano-structures and fluidic manipulation. We will also briefly discuss future directions and the current limitations and obstacles impeding even faster progress toward these goals.
authors.affiliation: Biomedical Engineering Department, Johns Hopkins University, Baltimore, MD 21218, USA. thwang@jhu.edu.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Friedrich
authors.affiliation: Mechanical Engineering Department, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Helena C
authors.initials: HC
authors.lastname: Zec
authors.affiliation: Biomedical Engineering Department, Johns Hopkins University, Baltimore, MD 21218, USA. thwang@jhu.edu and Mechanical Engineering Department, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Tza-Huei
authors.initials: TH
authors.lastname: Wang
mesh_terms: D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis*; D011173:Portraits as Topic
chemical_list: D009696:Nucleic Acids
references.citation: Nat Biotechnol. 2009 Nov;27(11):1013-23
references.pmid: 19898456
references.citation: Genome Res. 2014 Apr;24(4):688-96
references.pmid: 24418700
references.citation: J Clin Microbiol. 2000 Dec;38(12):4463-70
references.pmid: 11101581
references.citation: Gigascience. 2014 Jul 08;3:11
references.pmid: 25061512
references.citation: Sci Rep. 2015 Sep 03;5:13795
references.pmid: 26333806
references.citation: Nano Lett. 2005 Sep;5(9):1693-7
references.pmid: 16159207
references.citation: J Biophotonics. 2012 Aug;5(8-9):673-86
references.pmid: 22815200
references.citation: PLoS One. 2013;8(2):e55864
references.pmid: 23405223
references.citation: Anal Chem. 2003 Dec 15;75(24):6963-7
references.pmid: 14670059
references.citation: Nat Protoc. 2013 Mar;8(3):525-38
references.pmid: 23411634
references.citation: Am J Hum Genet. 1980 May;32(3):314-31
references.pmid: 6247908
references.citation: Nano Lett. 2005 Jul;5(7):1193-7
references.pmid: 16178209
references.citation: Anal Chem. 2013 Jan 15;85(2):1146-53
references.pmid: 23234458
references.citation: Nature. 2004 May 27;429(6990):457-63
references.pmid: 15164071
references.citation: Nat Biotechnol. 2012 Aug;30(8):771-6
references.pmid: 22797562
references.citation: Nucleic Acids Res. 2010 Oct;38(18):e177
references.pmid: 20699272
references.citation: Lab Chip. 2008 May;8(5):822-5
references.pmid: 18432356
references.citation: Nano Lett. 2010 Sep 8;10(9):3633-7
references.pmid: 20704324
references.citation: Proc Natl Acad Sci U S A. 2012 May 29;109(22):8477-82
references.pmid: 22586076
references.citation: Nat Biotechnol. 1996 Mar;14(3):303-8
references.pmid: 9630890
references.citation: Genomics Proteomics Bioinformatics. 2015 Feb;13(1):4-16
references.pmid: 25743089
references.citation: Lab Chip. 2009 Oct 7;9(19):2772-4
references.pmid: 19967112
references.citation: Nat Rev Cancer. 2011 Jun;11(6):426-37
references.pmid: 21562580
references.citation: Bioinformatics. 2015 Jun 15;31(12):i80-8
references.pmid: 26072512
references.citation: Biochim Biophys Acta. 2010 Jan;1805(1):105-17
references.pmid: 19931353
references.citation: Ann Clin Lab Sci. 1991 Jul-Aug;21(4):291-6
references.pmid: 1677556
references.citation: Genome Biol. 2013;14(9):R101
references.pmid: 24034426
references.citation: J Am Chem Soc. 2005 Apr 20;127(15):5354-9
references.pmid: 15826173
references.citation: ACS Nano. 2015 Jan 27;9(1):809-16
references.pmid: 25561163
references.citation: Nucleic Acids Res. 2002 Jun 15;30(12):e57
references.pmid: 12060695
references.citation: Anal Bioanal Chem. 2006 Aug;385(8):1384-8
references.pmid: 16802123
references.citation: PeerJ. 2015 Aug 27;3:e1225
references.pmid: 26336650
references.citation: Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6
references.pmid: 8790415
references.citation: Proc Natl Acad Sci U S A. 2014 Oct 21;111(42):15090-5
references.pmid: 25288749
references.citation: Appl Environ Microbiol. 1998 Oct;64(10):3748-53
references.pmid: 9758794
references.citation: Chem Rev. 2010 Aug 11;110(8):4910-47
references.pmid: 20394378
references.citation: N Engl J Med. 2011 Jan 6;364(1):33-42
references.pmid: 21142692
references.citation: Proc Natl Acad Sci U S A. 1971 Dec;68(12):2913-7
references.pmid: 4332003
references.citation: Clin Chem. 2013 Nov;59(11):1670-2
references.pmid: 24003063
references.citation: J Clin Microbiol. 2013 Apr;51(4):1066-70
references.pmid: 23363821
references.citation: PLoS One. 2013 Jun 21;8(6):e66493
references.pmid: 23805225
references.citation: Electrophoresis. 2013 Sep;34(17):2453-63
references.pmid: 23784786
references.citation: J Clin Oncol. 2014 Feb 20;32(6):579-86
references.pmid: 24449238
references.citation: Anal Biochem. 2013 Dec 15;443(2):261-8
references.pmid: 23994563
references.citation: N Engl J Med. 2012 Mar 8;366(10):883-892
references.pmid: 22397650
references.citation: Nat Methods. 2014 Mar;11(3):297-300
references.pmid: 24441935
references.citation: Biophys J. 2008 Sep 15;95(6):2964-75
references.pmid: 18515376
references.citation: Nat Biotechnol. 2002 Oct;20(10):1048-51
references.pmid: 12219075
references.citation: Biomol Detect Quantif. 2015 Mar;3:1-8
references.pmid: 26753127
references.citation: Genome Res. 2004 Jun;14(6):1137-46
references.pmid: 15173119
references.citation: Nat Methods. 2015 Apr;12(4):351-6
references.pmid: 25686389
references.citation: Langmuir. 2008 Sep 16;24(18):10524-31
references.pmid: 18683960
references.citation: Phys Rev Lett. 2002 Oct 21;89(17):178301
references.pmid: 12398707
references.citation: Nat Rev Genet. 2006 Feb;7(2):85-97
references.pmid: 16418744
references.citation: Proc Natl Acad Sci U S A. 2012 Feb 21;109(8):3065-70
references.pmid: 22315421
references.citation: Rev Sci Instrum. 2015 Jun;86(6):063702
references.pmid: 26133839
references.citation: Lab Chip. 2012 Feb 7;12(3):647-51
references.pmid: 22173785
references.citation: Lab Chip. 2012 Apr 21;12(8):1463-70
references.pmid: 22388662
references.citation: Science. 2003 Jan 31;299(5607):682-6
references.pmid: 12560545
references.citation: Proc Natl Acad Sci U S A. 2014 Apr 1;111(13):E1291-9
references.pmid: 24639501
references.citation: Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8583-8
references.pmid: 24843150
references.citation: Lab Chip. 2012 Sep 21;12(18):3341-7
references.pmid: 22842841
references.citation: Expert Rev Mol Diagn. 2014 Sep;14(7):787-801
references.pmid: 25109704
references.citation: Nucleic Acids Res. 2015 Oct 15;43(18):e117
references.pmid: 26019180
references.citation: Genome Biol. 2015 May 30;16:114
references.pmid: 26025440
references.citation: Proc Natl Acad Sci U S A. 2009 May 12;106(19):7702-7
references.pmid: 19380741
references.citation: BMC Genomics. 2007 Aug 15;8:278
references.pmid: 17697381
references.citation: Biomicrofluidics. 2014 Apr 16;8(2):024114
references.pmid: 24803963
references.citation: Anal Chem. 2003 Apr 1;75(7):1664-70
references.pmid: 12705600
references.citation: Nat Mater. 2005 Nov;4(11):826-31
references.pmid: 16379073
references.citation: Anal Chem. 2007 Oct 1;79(19):7576-9
references.pmid: 17715994
references.citation: Science. 2006 Dec 1;314(5804):1464-7
references.pmid: 17138901
references.citation: Front Microbiol. 2015 Aug 18;6:766
references.pmid: 26347715
references.citation: Electrophoresis. 2011 Feb;32(5):573-80
references.pmid: 21298673
references.citation: J Clin Microbiol. 2004 May;42(5):1965-76
references.pmid: 15131156
references.citation: Lab Chip. 2010 Apr 7;10(7):843-51
references.pmid: 20300670
references.citation: Lab Chip. 2011 Mar 7;11(5):863-73
references.pmid: 21249264
references.citation: DNA Repair (Amst). 2014 Aug;20:94-109
references.pmid: 24598576
references.citation: Gene. 1993 Dec 15;135(1-2):161-6
references.pmid: 8276254
references.citation: FEMS Microbiol Rev. 2009 Sep;33(5):892-916
references.pmid: 19453749
references.citation: BMC Genomics. 2015 Apr 11;16:286
references.pmid: 25886820
references.citation: Cell. 2007 Feb 23;128(4):683-92
references.pmid: 17320506
references.citation: Lab Chip. 2009 Aug 21;9(16):2348-55
references.pmid: 19636466
references.citation: Nat Nanotechnol. 2013 Oct;8(10):709-18
references.pmid: 24091454
references.citation: J Am Chem Soc. 2016 Jan 13;138(1):319-27
references.pmid: 26684193
references.citation: Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13294-9
references.pmid: 20616076
references.citation: Science. 1993 Oct 1;262(5130):110-4
references.pmid: 8211116
references.citation: Nanotechnology. 2014 Jul 11;25(27):275501
references.pmid: 24960034
references.citation: Lab Chip. 2013 Jan 21;13(2):240-51
references.pmid: 23202853
references.citation: Nature. 2008 May 1;453(7191):56-64
references.pmid: 18451855
references.citation: Proc Natl Acad Sci U S A. 2015 Jun 23;112(25):7689-94
references.pmid: 26056298
references.citation: Cancer Res. 2001 Feb 15;61(4):1659-65
references.pmid: 11245480
references.citation: Nano Lett. 2015 Aug 12;15(8):5641-6
references.pmid: 26156085
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):4893-8
references.pmid: 23479649
references.citation: Int J Mol Sci. 2013 Sep 13;14(9):18925-58
references.pmid: 24065096
references.citation: Cancer Res. 2003 Jul 15;63(14):3966-8
references.pmid: 12873992
references.citation: ACS Nano. 2015 Jan 27;9(1):290-300
references.pmid: 25569824
references.citation: Nucleic Acids Res. 2007;35(3):e16
references.pmid: 17175538
references.citation: Anal Chem. 2013 Feb 5;85(3):1932-9
references.pmid: 23331316
references.citation: Nucleic Acids Res. 2015 Dec 15;43(22):e154
references.pmid: 26304549
references.citation: Nucleic Acids Res. 2010 Aug;38(15):e159
references.pmid: 20571086
references.citation: Clin Chem. 2015 Oct;61(10):1305-6
references.pmid: 26286915
references.citation: Hum Immunol. 2015 Dec;76(12):891-6
references.pmid: 26028281
references.citation: Biophys J. 2008 Jul;95(1):273-86
references.pmid: 18339746
references.citation: J Clin Microbiol. 2012 Sep;50(9):3063-5
references.pmid: 22718933
references.citation: Nat Biotechnol. 2013 Feb;31(2):135-41
references.pmid: 23263233
references.citation: Nat Nanotechnol. 2009 Apr;4(4):265-70
references.pmid: 19350039
references.citation: Science. 1997 Jun 27;276(5321):2016-21
references.pmid: 9197259
references.citation: Angew Chem Int Ed Engl. 2010;49(3):556-9
references.pmid: 20014084
references.citation: Diagn Microbiol Infect Dis. 2014 Dec;80(4):285-6
references.pmid: 25277746
references.citation: N Engl J Med. 2003 Nov 20;349(21):2042-54
references.pmid: 14627790
references.citation: Genome Med. 2015 Sep 29;7:99
references.pmid: 26416663
references.citation: Curr Opin Biotechnol. 2014 Feb;25:69-77
references.pmid: 24484883
references.citation: Nature. 2015 Jan 29;517(7536):608-11
references.pmid: 25383537
references.citation: J Am Chem Soc. 2010 Apr 28;132(16):5793-8
references.pmid: 20364832
references.citation: Nano Lett. 2014 Aug 13;14(8):4729-35
references.pmid: 25054542
references.citation: Clin Chem. 2009 Dec;55(12):2121-9
references.pmid: 19815611
references.citation: Genome Res. 2001 Sep;11(9):1584-93
references.pmid: 11544203
references.citation: Anal Chem. 1997 Oct 1;69(19):3915-20
references.pmid: 9322430
references.citation: Phys Rev Lett. 2015 May 15;114(19):198303
references.pmid: 26024203
references.citation: Sci Rep. 2013;3:1389
references.pmid: 23474808
references.citation: Curr Opin Microbiol. 2015 Feb;23:110-20
references.pmid: 25461581
references.citation: Cancer Res. 2015 Jul 1;75(13):2587-93
references.pmid: 26069246
references.citation: Int J Mol Sci. 2011;12(8):5135-56
references.pmid: 21954349
references.citation: Nat Biotechnol. 2008 Oct;26(10):1146-53
references.pmid: 18846088
references.citation: Lab Chip. 2010 Jun 21;10(12):1543-9
references.pmid: 20358051
references.citation: Lab Chip. 2012 Mar 21;12(6):1094-101
references.pmid: 22298224
references.citation: Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10012-6
references.pmid: 16000405
references.citation: Biomicrofluidics. 2011 Sep;5(3):34106-341068
references.pmid: 21869910
references.citation: Lab Chip. 2010 Jan 21;10(2):161-4
references.pmid: 20066242
references.citation: Chem Rev. 2013 Apr 10;113(4):2584-667
references.pmid: 23140825
references.citation: PLoS One. 2013 Apr 17;8(4):e61762
references.pmid: 23613926
references.citation: Anal Bioanal Chem. 2005 Aug;382(8):1771-82
references.pmid: 16075229
references.citation: Nat Methods. 2011 Nov 20;9(1):75-7
references.pmid: 22101853
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6264-8
references.pmid: 8692803
references.citation: BMC Genomics. 2014 May 20;15:387
references.pmid: 24885025
references.citation: Genome Biol. 2013 Jul 03;14(7):405
references.pmid: 23822731
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3670-5
references.pmid: 10097095
references.citation: Genome Biol. 2015 Sep 30;16:204
references.pmid: 26420219
references.citation: Lab Chip. 2014 Oct 7;14(19):3881-93
references.pmid: 25133764
references.citation: J Am Chem Soc. 2011 May 11;133(18):6898-901
references.pmid: 21504160
references.citation: BMC Res Notes. 2014 Oct 08;7:704
references.pmid: 25297888
references.citation: Nat Methods. 2010 Jan;7(1):47-9
references.pmid: 19946276
references.citation: Nano Lett. 2012 Jul 11;12(7):3861-6
references.pmid: 22698062
references.citation: Nat Biotechnol. 2008 Mar;26(3):317-25
references.pmid: 18278033
references.citation: Nat Methods. 2015 Aug;12(8):780-6
references.pmid: 26121404
references.citation: J Virol Methods. 2012 Jan;179(1):45-50
references.pmid: 21983150
references.citation: N Engl J Med. 2011 Aug 25;365(8):709-17
references.pmid: 21793740
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2673-8
references.pmid: 17296933
references.citation: Anal Chem. 2014 May 6;86(9):4157-63
references.pmid: 24734940
references.citation: J Clin Diagn Res. 2015 Aug;9(8):DC24-7
references.pmid: 26435946
references.citation: PLoS Genet. 2009 Nov;5(11):e1000711
references.pmid: 19936062
references.citation: Nat Methods. 2006 Jan;3(1):41-6
references.pmid: 16369552
references.citation: Trends Mol Med. 2012 Jul;18(7):405-16
references.pmid: 22727782
references.citation: Methods. 2015 Jan 15;72:41-50
references.pmid: 25204781
references.citation: Analyst. 2014 Jun 21;139(12):2968-81
references.pmid: 24770716
references.citation: Nature. 2012 Jul 19;487(7407):320-4
references.pmid: 22763444
references.citation: Gigascience. 2015 Aug 04;4:35
references.pmid: 26244089
references.citation: Genetics. 2016 Jan;202(1):351-62
references.pmid: 26510793
references.citation: Nat Protoc. 2013 Aug;8(8):1459-74
references.pmid: 23845960
references.citation: Nat Methods. 2015 Aug;12(8):733-5
references.pmid: 26076426
references.citation: Lab Chip. 2009 Apr 21;9(8):1065-72
references.pmid: 19350088
references.citation: ACS Nano. 2012 Sep 25;6(9):7928-34
references.pmid: 22816516
references.citation: Nat Methods. 2010 Jun;7(6):461-5
references.pmid: 20453866
references.citation: Theranostics. 2012;2(7):631-54
references.pmid: 22916072
references.citation: Electrophoresis. 2011 Feb;32(5):581-7
references.pmid: 21290387
references.citation: Nat Biotechnol. 2012 Jul 01;30(7):693-700
references.pmid: 22750884
references.citation: Nanomedicine. 2005 Jun;1(2):115-21
references.pmid: 17292066
references.citation: Nature. 2007 May 24;447(7143):433-40
references.pmid: 17522677
references.citation: Anal Biochem. 2010 Jul 1;402(1):83-90
references.pmid: 20307487
references.citation: Anal Chem. 2010 Mar 15;82(6):2480-7
references.pmid: 20184350
references.citation: Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3178-9
references.pmid: 25733911
references.citation: Anal Chem. 2013 Jul 16;85(14):6855-9
references.pmid: 23782428
references.citation: Nature. 1985 Mar 7-13;314(6006):67-73
references.pmid: 3856104
references.citation: Future Microbiol. 2007 Apr;2(2):175-85
references.pmid: 17661654
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10848-53
references.pmid: 20534489
references.citation: Nat Methods. 2011 Mar;8(3):242-5
references.pmid: 21297618
references.citation: Anal Chem. 2004 Sep 15;76(18):5293-301
references.pmid: 15362885
references.citation: Nat Biotechnol. 2015 Mar;33(3):296-300
references.pmid: 25485618
references.citation: Anal Biochem. 2012 Jul 1;426(1):13-21
references.pmid: 22484041
references.citation: Opt Lett. 1986 May 1;11(5):288
references.pmid: 19730608
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):10979-83
references.pmid: 15252203
references.citation: J Antimicrob Chemother. 2015 Oct;70(10):2775-8
references.pmid: 26221019
references.citation: Lab Chip. 2011 Feb 7;11(3):435-42
references.pmid: 21072428
references.citation: mBio. 2014 Sep 09;5(5):e01377-14
references.pmid: 25205090
references.citation: Science. 1994 Sep 30;265(5181):2096-8
references.pmid: 7522347
references.citation: PCR Methods Appl. 1994 Feb;3(4):S65-75
references.pmid: 8173510
references.citation: Chem Commun (Camb). 2011 Apr 7;47(13):3717-35
references.pmid: 21283891
references.citation: Sci Rep. 2015 Jul 07;5:11996
references.pmid: 26149338
references.citation: Anal Chem. 2011 Nov 15;83(22):8604-10
references.pmid: 22035192
references.citation: Gigascience. 2014 Dec 30;3(1):34
references.pmid: 25671094
references.citation: Lab Chip. 2011 May 21;11(10):1721-9
references.pmid: 21431167
references.citation: Science. 2000 May 12;288(5468):1026-9
references.pmid: 10807568
references.citation: Science. 2009 Jan 2;323(5910):133-8
references.pmid: 19023044
references.citation: Nat Mater. 2005 Jun;4(6):435-46
references.pmid: 15928695
references.citation: Lab Chip. 2009 Aug 21;9(16):2286-92
references.pmid: 19636458
references.citation: Proc Natl Acad Sci U S A. 2013 May 7;110(19):7772-7
references.pmid: 23610441