Droplet microfluidics for high‐sensitivity and high‐throughput detection and screening of disease biomarkers

Advanced Review

Tza‐Huei Wang, Kuangwen Hsieh, Aniruddha M. Kaushik

Published Online: May 24 2018

DOI: 10.1002/wnan.1522

Full article on Wiley Online Library: HTML PDF

Can't access this content? Tell your librarian.

Biomarkers are nucleic acids, proteins, single cells, or small molecules in human tissues or biological fluids whose reliable detection can be used to confirm or predict disease and disease states. Sensitive detection of biomarkers is therefore critical in a variety of applications including disease diagnostics, therapeutics, and drug screening. Unfortunately for many diseases, low abundance of biomarkers in human samples and low sample volumes render standard benchtop platforms like 96‐well plates ineffective for reliable detection and screening. Discretization of bulk samples into a large number of small volumes (fL‐nL) via droplet microfluidic technology offers a promising solution for high‐sensitivity and high‐throughput detection and screening of biomarkers. Several microfluidic strategies exist for high‐throughput biomarker digitization into droplets, and these strategies have been utilized by numerous droplet platforms for nucleic acid, protein, and single‐cell detection and screening. While the potential of droplet‐based platforms has led to burgeoning interest in droplets, seamless integration of sample preparation technologies and automation of platforms from biological sample to answer remain critical components that can render these platforms useful in the clinical setting in the near future. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease

Discretization facilitates high‐throughput, high‐sensitivity, rapid, and quantitative analysis for rare biomarkers in a sample. For effective detection of a biomarker of interest, conventional bulk analysis (left) is restricted to a few replicate reactions between microliters and milliliters each, wherein signal can be drowned out by high concentration of sample background, limiting overall sensitivity and speed. In contrast, digitization of sample (right) into femtoliter to nanoliter volume droplets facilitates background reduction and subsequently greater sensitivity and speed. Furthermore, encapsulation of single targets into these droplets allow for absolute quantification of rare targets

[ Normal View | Magnified View ]

Increasing multiplexing and screening capacity of droplet platforms. (a) A droplet‐on‐demand system was developed to interface droplet chips with off‐chip reservoirs in order to construct libraries of droplets with controllable input concentrations of sample/reagents and controllable volumes at 30 Hz. (Reprinted with permission from Churski et al. (). Copyright 2010 The Royal Society of Chemistry) (b) A device was developed to facilitate the transition of nanoliter plugs that were constructed with controlled combinations of sample/reagents, into picoliter droplets without compromising droplet stability and uniformity. (Reprinted with permission from P. Zhang, Kaushik, Hsieh, and Wang (). Copyright 2017 IEEE). (c) A serial sample loading (SSL) system was developed to interface droplet platforms with conventional 96‐well plates to facilitate automated sample and reagent loading. (Reprinted with permission from T. D. Rane, Zec, and Wang (). Copyright 2012 Sage Publishing)

[ Normal View | Magnified View ]

Droplet based bacterial growth quantification and antibiotic susceptibility testing (AST). (a) A ddCFU platform was developed to rapidly count viable bacteria within a sample. Critically, this platform followed a fragmented workflow that necessitated separate modules for (i) droplet generation and encapsulation of single bacteria, (ii) off‐chip bacterial incubation, and (iii) droplet detection. (Reprinted with permission from O. Scheler et al. (). Copyright 2017 The Royal Society of Chemistry). (b) A similar workflow was used to develop a droplet‐based AST assay. After coencapsulation of single cells of Staphylococcus aureus with blood plasma and ampicillin into 4‐nL droplets, the pathogen's susceptibility/resistance to the drug was determined in 7.5 hr. (Reprinted with permission from Boedicker et al. (). Copyright 2008 The Royal Society of Chemistry). (c) An integrated one‐step platform was developed for bacterial growth analysis and antibiotic susceptibility testing (Kaushik et al., ). Critically, the use of much smaller 20‐pL droplets in this platform enabled detection of single‐cell E. coli growth and its susceptibility/resistance to gentamicin in as little as 1 hr, equivalent to two to three replications of the bacterial cell. (Reprinted with permission from Kaushik et al. (). Copyright 2017 Elsevier)

[ Normal View | Magnified View ]

Droplet based detection of single‐cell biomarkers. (a) An integrated amplification‐free platform for genetic detection of pathogens was developed. Single cells of E. coli were coencapsulated with peptide nucleic acid beacons, complimentary to a specific region within the 16S rRNA of the pathogen. Following on‐chip thermal lysis, rRNA release, and probe hybridization, droplet fluorescence was detected and used to quantify pathogen load within a sample. (Reprinted with permission from T. D. Rane, Zec, Puleo, et al. (). Copyright 2012 The Royal Society of Chemistry). (b) An enzyme‐tagged antibody was used to detect the typically low‐abundance cell‐surface protein biomarker CCR5 (a coreceptor in HIV‐1 infection) in U937 cells. (Reprinted with permission from Joensson et al. (). Copyright 2009 John Wiley and Sons)

[ Normal View | Magnified View ]

Droplet based detection of protein biomarkers. (a) A bead‐based sandwich assay, similar to enzyme‐linked immunosorbent assay (ELISA), was developed for quantifying PSA, a prostate cancer biomarker, in buffer (Shim et al., ). The target protein is tagged with a reporter enzyme and hybridized to the bead. Beads and substrate are coflown to generate femtoliter droplets. After 10 min of incubation, this platform is able to detect protein biomarker concentrations as low as 46 fM. (Reprinted with permission from Shim et al. ()). Copyright 2013 American Chemical Society). (b) Matrix metalloproteinase (MMP) biomarkers secreted into the PF of clinical endometriosis tissue sample were combined with a droplet library of four FRET‐based protease substrates with and without specific inhibitors and incubated on chip. Following at least 3 hr of fluorescence monitoring, protease activity matrix analysis (PrAMA) was conducted to assay protease activity within droplets. (Reprinted with permission from Chen et al. (). Copyright 2013 American Chemical Society). (c) In order to scale up PrAMA analysis to a larger set of MMPs and substrates, a high‐throughput combinatorial valve‐based platform was developed (T. D. Rane et al., ). A 650 unique combinations of MMPs and substrates were screened in continuous flow following 12 min incubation using this approach

[ Normal View | Magnified View ]

Amplification‐free quantification of RNA using droplet digital enzyme‐linked oligonucleotide hybridization assay (ELOHA) (Reprinted with permission from Guan et al. ()). Copyright 2015 Creative Commons Attribution 4.0 License). (a) A sandwiched complex consisting of capture oligo‐coated magnetic beads hybridized to a single molecule of target RNA which is then hybridized to an enzyme‐linked detection oligo is coflowed into droplets along with corresponding enzyme substrate. Following in‐line incubation, droplets containing single RNA exhibit strong fluorescence, whereas droplets without the sandwiched complex exhibit weak fluorescence. (b) By interrogating ~10⁶ droplets, the percentage of highly fluorescent droplets can be used to quantify input concentration of 16S rRNA from N. gonorrhoeae down to 600 molecule copies in 100 μL of sample

[ Normal View | Magnified View ]

Droplet‐based detection of nucleic acid biomarkers. (a) Droplet‐based digital PCR was used to screen for mutations in the KRAS oncogene. (Reprinted with permission from Pekin et al. ()). Copyright 2011 The Royal Society of Chemistry). TaqMan probes specific for the wild‐type and mutant genes were encapsulated in droplets that contain at most one haploid genome. The emulsion was then thermocycled off chip, and reinjected for fluorescence detection. By optically coding droplet groups, six distinct mutations in KRAS were detected simultaneously using this platform. (B) An integrated platform for viral RNA detection using reverse transcription polymerase chain reaction (RT‐PCR) was developed, where 70‐pL droplets containing MS2 virions and RT‐PCR reagents were immobilized in microfluidic channels. An integrated thermal cycler provided temperatures necessary for reverse transcription and PCR, and a CCD camera was used for fluorescence detection. (Reprinted with permission from Beer et al. (). Copyright 2007 American Chemical Society). (C) Loop‐mediated isothermal amplification was demonstrated for detection and quantification of N. gonorrhoeae gDNA down to 600 copies per μL. Critically, the assay was conducted in continuous flow in an integrated platform that facilitated digitization of targets, on‐chip incubation, and detection. (Reprinted with permission from Rane et al. (). Copyright 2014 The Royal Society of Chemistry)

[ Normal View | Magnified View ]

Fragmented and integrated droplet platforms. (a) Most droplet platforms utilize a separate device for (i) droplet generation, (ii) a thermocycler or incubator for droplet incubation, and (iii) another device for droplet detection. (Reprinted with permission from Pekin et al. (). Copyright 2011 The Royal Society of Chemistry). (b) Imaging‐based integrated platforms use (i) a single device for droplet generation and incubation. Droplets are incubated in an on‐chip droplet reservoir that rests on a heat block and (ii) real‐time imaging or microscopy may be used to observe these droplets. (Reprinted with permission from Hatch, Fisher, Tovar, et al. (). Copyright 2011 The Royal Society of Chemistry). (C) Integrated platforms that facilitate continuous‐flow detection of droplets typically contain (i) delay lines for on‐chip droplet incubation. Often, these delay lines may contain constrictions to reduce variability in droplet speed through the incubation region. (Reprinted with permission from Frenz et al. (). Copyright 2009 The Royal Society of Chemistry). These constrictions also serve as points for droplet detection where individual droplets can be sequentially measured. (ii) Integrated continuous flow platforms may feature a Peltier heater to heat the incubation delay line and a laser excitation source and avalanche photodiode (APD) detector for continuous measurements of droplet fluorescence. (Reprinted with permission from Kaushik et al. (). Copyright 2017 Elsevier)

[ Normal View | Magnified View ]

Methods for high‐throughput droplet generation. (a) Valve actuation involves pneumatically pulsing polydimethylsiloxane (PDMS) “quake” valves to generate droplets. They offer greater control of droplet size, content, and motion, but are limited in generation speed and throughput (Reprinted with permission from Guo et al. (). Copyright 2010 AIP publishing). (b) Cross‐flow devices feature a “T‐junction” where an aqueous stream meets a flowing continuous phase to generate droplets. (Reprinted with permission from Zagnoni, Anderson, and Cooper (). Copyright 2010 American Chemical Society). (c) Flow‐focusing devices are most commonly used for droplet generation and feature a junction where a flowing aqueous stream is sheared by two perpendicularly intersecting streams of the continuous phase (Hindson et al., ). (d) Step‐emulsification devices feature a three dimensional (3D) step where an aqueous stream enters into a much larger oil reservoir, creating a droplet. (Reprinted with permission from R. Dangla, Kayi, and Baroud ()). Copyright 2012 National Academy of Sciences). Step emulsifiers may be parallelized to generate droplets at very high speeds, but are more difficult to control in content and movement

[ Normal View | Magnified View ]

References

Akbari,, S., & Pirbodaghi,, T. (2014). A droplet‐based heterogeneous immunoassay for screening single cells secreting antigen‐specific antibodies. Lab on a Chip, 14(17), 3275–3280. https://doi.org/10.1039/C4LC00082J
Anna,, S. L., Bontoux,, N., & Stone,, H. A. (2003). Formation of dispersions using “flow focusing” in microchannels. Applied Physics Letters, 82(3), 364–366. https://doi.org/10.1063/1.1537519
Arayanarakool,, R., Shui,, L., Kengen,, S. W. M., van den Berg,, A., & Eijkel,, J. C. T. (2013). Single‐enzyme analysis in a droplet‐based micro‐ and nanofluidic system. Lab on a Chip, 13(10), 1955–1962. https://doi.org/10.1039/c3lc41100a
Aronson,, J. K. (2005). Biomarkers and surrogate endpoints. British Journal of Clinical Pharmacology, 59(5), 491–494. https://doi.org/10.1111/j.1365-2125.2005.02435.x
Axt,, B., Hsieh,, Y., Nalayanda,, D., & Wang,, T. (2017). Impedance feedback control of microfluidic valves for reliable post processing combinatorial droplet injection. Biomedical Microdevices, 1–9. https://doi.org/10.1007/s10544-017-0203-2
Baker,, M. (2012). Digital PCR hits its stride. Nature Methods, 9(6), 541–544. https://doi.org/10.1038/nmeth.2027
Baret,, J. C., Beck,, Y., Billas‐Massobrio,, I., Moras,, D., & Griffiths,, A. D. (2010). Quantitative cell‐based reporter gene assays using droplet‐based microfluidics. Chemistry and Biology, 17(5), 528–536. https://doi.org/10.1016/j.chembiol.2010.04.010
Beer,, N. R., Hindson,, B. J., Wheeler,, E. K., Hall,, S. B., Rose,, K. A., Kennedy,, I. M., & Colston,, B. W. (2007). On‐chip, real‐time, single‐copy polymerase chain reaction in picoliter droplets. Analytical Chemistry, 79(22), 8471–8475. https://doi.org/10.1021/ac701809w
Beer,, N. R., Wheeler,, E. K., Lee‐Houghton,, L., Watkins,, N., Nasarabadi,, S., Hebert,, N., … Colston,, B. W. (2008). On‐chip single‐copy real‐time reverse‐transcription PCR in isolated picoliter droplets. Analytical Chemistry, 80(6), 1854–1858. https://doi.org/10.1021/ac800048k
Belgrader,, P., Tanner,, S. C., Regan,, J. F., Koehler,, R., Hindson,, B. J., & Brown,, A. S. (2013). Droplet digital PCR measurement of HER2 copy number alteration in formalin‐fixed paraffin‐embedded breast carcinoma tissue. Clinical Chemistry, 59(6), 991–994. https://doi.org/10.1373/clinchem.2012.197855
Beltrame,, L., Di Marino,, M., Fruscio,, R., Calura,, E., Chapman,, B., Clivio,, L., … Marchini,, S. (2015). Profiling cancer gene mutations in longitudinal epithelial ovarian cancer biopsies by targeted next‐generation sequencing: A retrospective study. Annals of Oncology, 26(7), 1363–1371. https://doi.org/10.1093/annonc/mdv164
Bian,, X., Jing,, F., Li,, G., Fan,, X., Jia,, C., Zhou,, H., … Zhao,, J. (2015). A microfluidic droplet digital PCR for simultaneous detection of pathogenic Escherichia coli O157 and Listeria monocytogenes. Biosensors and Bioelectronics, 74, 770–777. https://doi.org/10.1016/j.bios.2015.07.016
Boedicker,, J. Q., Li,, L., Kline,, T. R., & Ismagilov,, R. F. (2008). Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug‐based microfluidics. Lab on a Chip, 8(8), 1265–1272. https://doi.org/10.1039/b804911d
Boehme,, C. C., Nabeta,, P., Hillemann,, D., Nicol,, M. P., Shenai,, S., Krapp,, F., … Perkins,, M. D. (2010). Rapid molecular detection of tuberculosis and rifampin resistance. The New England Journal of Medicine, 363(11), 1005–1015. https://doi.org/10.1056/NEJMoa0907847
Boitard,, L., Cottinet,, D., Kleinschmitt,, C., Bremond,, N., Baudry,, J., Yvert,, G., & Bibette,, J. (2012). Monitoring single‐cell bioenergetics via the coarsening of emulsion droplets. Proceedings of the National Academy of Sciences, 109(19), 7181–7186. https://doi.org/10.1073/pnas.1200894109
Breadmore,, M. C., Wolfe,, K. A., Arcibal,, I. G., Leung,, W. K., Dickson,, D., Giordano,, B. C., … Landers,, J. P. (2003). Microchip‐based purification of DNA from biological samples. Analytical Chemistry, 75(8), 1880–1886. https://doi.org/10.1021/ac0204855
Brunetto,, G. S., Massoud,, R., Leibovitch,, E. C., Caruso,, B., Johnson,, K., Ohayon,, J., … Jacobson,, S. (2014). Digital droplet PCR (ddPCR) for the precise quantification of human T‐lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations. Journal of Neurovirology, 20(4), 341–351. https://doi.org/10.1007/s13365-014-0249-3
Cecchini,, M. P., Hong,, J., Lim,, C., Choo,, J., Albrecht,, T., DeMello,, A. J., & Edel,, J. B. (2011). Ultrafast surface enhanced resonance Raman scattering detection in droplet‐based microfluidic systems. Analytical Chemistry, 83(8), 3076–3081. https://doi.org/10.1021/ac103329b
Chang‐Hao Tsao,, S., Weiss,, J., Hudson,, C., Christophi,, C., Cebon,, J., Behren,, A., & Dobrovic,, A. (2015). Monitoring response to therapy in melanoma by quantifying circulating tumour DNA with droplet digital PCR for BRAF and NRAS mutations. Scientific Reports, 5(1), 11198. https://doi.org/10.1038/srep11198
Chen,, C. H., Miller,, M. A., Sarkar,, A., Beste,, M. T., Isaacson,, K. B., Lauffenburger,, D. A., … Han,, J. (2013). Multiplexed protease activity assay for low‐volume clinical samples using droplet‐based microfluidics and its application to endometriosis. Journal of the American Chemical Society, 135(5), 1645–1648. https://doi.org/10.1021/ja307866z
Chen,, C. H., Sarkar,, A., Song,, Y. A., Miller,, M. A., Kim,, S. J., Griffith,, L. G., … Han,, J. (2011). Enhancing protease activity assay in droplet‐based microfluidics using a biomolecule concentrator. Journal of the American Chemical Society, 133(27), 10368–10371. https://doi.org/10.1021/ja2036628
Cho,, S. K., Moon,, H., & Kim,, C. J. (2003). Creating, transporting, cutting, and merging liquid droplets by electrowetting‐based actuation for digital microfluidic circuits. Journal of Microelectromechanical Systems, 12(1), 70–80. https://doi.org/10.1109/JMEMS.2002.807467
Choi,, K., Ng,, A. H. C., Fobel,, R., & Wheeler,, A. R. (2012). Digital microfluidics. Annual Review of Analytical Chemistry, 5(1), 413–440. https://doi.org/10.1146/annurev-anchem-062011-143028
Chou,, W. L., Lee,, P. Y., Yang,, C. L., Huang,, W. Y., & Lin,, Y. S. (2015). Recent advances in applications of droplet microfluidics. Micromachines, 6, 1249–1271. https://doi.org/10.3390/mi6091249
Chrimes,, A. F., Khoshmanesh,, K., Stoddart,, P. R., Mitchell,, A., & Kalantar‐zadeh,, K. (2013). Microfluidics and Raman microscopy: Current applications and future challenges. Chemical Society Reviews, 42(13), 5880–5906. https://doi.org/10.1039/c3cs35515b
Churski,, K., Korczyk,, P., & Garstecki,, P. (2010). High‐throughput automated droplet microfluidic system for screening of reaction conditions. Lab on a Chip, 10(7), 816–818. https://doi.org/10.1039/b925500a
Clausell‐Tormos,, J., Lieber,, D., Baret,, J. C., El‐Harrak,, A., Miller,, O. J., Frenz,, L., … Merten,, C. A. (2008). Droplet‐based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms. Chemistry and Biology, 15(5), 427–437. https://doi.org/10.1016/j.chembiol.2008.04.004
Clinical Laboratory Standards Institute. (2014). Performance standards for antimicrobial susceptibility testing; Twenty‐Fourth Informational Supplement (Vol. 32). Wayne, PA: Author.
Collins,, D. J., Neild,, A., deMello,, A., Liu,, A.‐Q., & Ai,, Y. (2015). The Poisson distribution and beyond: Methods for microfluidic droplet production and single cell encapsulation. Lab on a Chip, 15, 3439–3459. https://doi.org/10.1039/C5LC00614G
Cristobal,, G., Arbouet,, L., Sarrazin,, F., Talaga,, D., Bruneel,, J.‐L., Joanicot,, M., & Servant,, L. (2006). On‐line laser Raman spectroscopic probing of droplets engineered in microfluidic devices. Lab on a Chip, 6(9), 1140–1146. https://doi.org/10.1039/b602702d
Crowley,, E., Di Nicolantonio,, F., Loupakis,, F., & Bardelli,, A. (2013). Liquid biopsy: Monitoring cancer‐genetics in the blood. Nature Reviews. Clinical Oncology, 10(8), 472–484. https://doi.org/10.1038/nrclinonc.2013.110
Dangla,, R., Fradet,, E., Lopez,, Y., & Baroud,, C. N. (2013). The physical mechanisms of step emulsification. Journal of Physics D: Applied Physics, 46(11), 114003. https://doi.org/10.1088/0022-3727/46/11/114003
Dangla,, R., Kayi,, S. C., & Baroud,, C. N. (2013). Droplet microfluidics driven by gradients of confinement. Proceedings of the National Academy of Sciences, 110(3), 853–858. https://doi.org/10.1073/pnas.1209186110
Davenport,, M., Mach,, K. E., Shortliffe,, L. M. D., Banaei,, N., Wang,, T.‐H., & Liao,, J. C. (2017). New and developing diagnostic technologies for urinary tract infections. Nature Reviews Urology, 14, 296–310. https://doi.org/10.1038/nrurol.2017.20
De Angelis,, G., Rittenhouse,, H. G., Mikolajczyk,, S. D., Blair Shamel,, L., & Semjonow,, A. (2007). Twenty years of PSA: From prostate antigen to tumor marker. Reviews in Urology, 9(3), 113–123. Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/17934568\nhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC2002501
De Mattos‐Arruda,, L., Mayor,, R., Ng,, C. K. Y., Weigelt,, B., Martínez‐Ricarte,, F., Torrejon,, D., … Seoane,, J. (2015). Cerebrospinal fluid‐derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nature Communications, 6, 8839. https://doi.org/10.1038/ncomms9839
Devonshire,, A. S., Honeyborne,, I., Gutteridge,, A., Whale,, A. S., Nixon,, G., Wilson,, P., … Huggett,, J. F. (2015). Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR. Analytical Chemistry, 87(7), 3706–3713. https://doi.org/10.1021/ac5041617
DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents. (2016). Guidelines for the use of antiretroviral agents in HIV‐1‐infected adults and adolescents. Retrieved from http://aidsinfo.nih.gov/guidelines
Ding,, Y., Casadevall i Solvas,, X., & deMello,, A. (2015). “V‐junction”: A novel structure for high‐speed generation of bespoke droplet flows. The Analyst, 140(2), 414–421. https://doi.org/10.1039/C4AN01730G
Doyle,, T., Smith,, C., Vitiello,, P., Cambiano,, V., Johnson,, M., Owen,, A., … Geretti,, A. M. (2014). The importance of HIV RNA detection below 50 copies per mL in HIV‐positive patients on antiretroviral therapy: An observational study. The Lancet, 383, S44. https://doi.org/10.1016/S0140-6736(14)60307-X
Dressman,, D., Yan,, H., Traverso,, G., Kinzler,, K. W., & Vogelstein,, B. (2003). Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proceedings of the National Academy of Sciences of the United States of America, 100(15), 8817–8822. https://doi.org/10.1073/pnas.1133470100
Dreyfus,, R., Tabeling,, P., & Willaime,, H. (2003). Ordered and disordered patterns in two‐phase flows in microchannels. Physical Review Letters, 90(14), 144505. https://doi.org/10.1103/PhysRevLett.90.144505
Dutka,, F., Opalski,, A. S., & Garstecki,, P. (2016). Nano‐liter droplet libraries from a pipette: Step‐emulsificator that stabilizes droplet volume against variation in flow rate. Lab on a Chip, 16(11), 2044–2049. https://doi.org/10.1039/C6LC00265J
Easley,, C. J., Karlinsey,, J. M., Bienvenue,, J. M., Legendre,, L. a., Roper,, M. G., Feldman,, S. H., … Landers,, J. P. (2006). A fully integrated microfluidic genetic analysis system with sample‐in‐answer‐out capability. Proceedings of the National Academy of Sciences of the United States of America, 103(51), 19272–19277. https://doi.org/10.1073/pnas.0604663103
Eastburn,, D. J., Sciambi,, A., & Abate,, A. R. (2013). Ultrahigh‐throughput mammalian single‐cell reverse‐transcriptase polymerase chain reaction in microfluidic drops. Analytical Chemistry, 85(16), 8016–8021. http://doi.org/10.1021/ac402057q
Ellington,, A. A., Kullo,, I. J., Bailey,, K. R., & Klee,, G. G. (2010). Antibody‐based protein multiplex platforms: Technical and operational challenges. Clinical Chemistry, 56, 186–193. https://doi.org/10.1373/clinchem.2009.127514
Frenz,, L., Blank,, K., Brouzes,, E., & Griffiths,, A. D. (2009). Reliable microfluidic on‐chip incubation of droplets in delay‐lines. Lab on a Chip, 9(10), 1344–1348. https://doi.org/10.1039/B816049J
Garnett,, M. J., Edelman,, E. J., Heidorn,, S. J., Greenman,, C. D., Dastur,, A., Lau,, K. W., … Benes,, C. H. (2012). Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature, 483(7391), 570–575. https://doi.org/10.1038/nature11005
Garstecki,, P., Fuerstman,, M. J., Stone,, H. A., & Whitesides,, G. M. (2006). Formation of droplets and bubbles in a microfluidic T‐junction—scaling and mechanism of break‐up. Lab on a Chip, 6(3), 437. http://doi.org/10.1039/b510841a
Gu,, S., Lu,, Y., Ding,, Y., Li,, L., Song,, H., Wang,, J., & Wu,, Q. (2014). A droplet‐based microfluidic electrochemical sensor using platinum‐black microelectrode and its application in high sensitive glucose sensing. Biosensors and Bioelectronics, 55, 106–112. https://doi.org/10.1016/j.bios.2013.12.002
Guan,, W., Chen,, L., Rane,, T. D., & Wang,, T.‐H. (2015). Droplet digital enzyme‐linked oligonucleotide hybridization assay for absolute RNA quantification. Scientific Reports, 5(April), 13795. https://doi.org/10.1038/srep13795
Guo,, F., Liu,, K., Ji,, X. H., Ding,, H. J., Zhang,, M., Zeng,, Q., … Zhao,, X. Z. (2010). Valve‐based microfluidic device for droplet on‐demand operation and static assay. Applied Physics Letters, 97(23), 233701. https://doi.org/10.1063/1.3521283
Gupta,, A., Matharoo,, H. S., Makkar,, D., & Kumar,, R. (2014). Droplet formation via squeezing mechanism in a microfluidic flow‐focusing device. Computers and Fluids, 100, 218–226. https://doi.org/10.1016/j.compfluid.2014.05.023
Guttenberg,, Z., Müller,, H., Habermüller,, H., Geisbauer,, A., Pipper,, J., Felbel,, J., … Wixforth,, A. (2005). Planar chip device for PCR and hybridization with surface acoustic wave pump. Lab on a Chip, 5(3), 308–317. https://doi.org/10.1039/B412712A
Guttery,, D. S., Page,, K., Hills,, A., Woodley,, L., Marchese,, S. D., Rghebi,, B., … Shaw,, J. A. (2015). Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor‐positive metastatic breast cancer. Clinical Chemistry, 61(7), 974–982. https://doi.org/10.1373/clinchem.2015.238717
Han,, Z., Li,, W., Huang,, Y., & Zheng,, B. (2009). Measuring rapid enzymatic kinetics by electrochemical method in droplet‐based microfluidic devices with pneumatic valves. Analytical Chemistry, 81(14), 5840–5845. https://doi.org/10.1021/ac900811y
Hanash,, S. M., Baik,, C. S., & Kallioniemi,, O. (2011). Emerging molecular biomarkers‐blood‐based strategies to detect and monitor cancer. Nature Reviews Clinical Oncology, 8, 142–150. https://doi.org/10.1038/nrclinonc.2010.220
Hatch,, A. C., Fisher,, J. S., Pentoney,, S. L., Yang,, D. L., & Lee,, A. P. (2011). Tunable 3D droplet self‐assembly for ultra‐high‐density digital micro‐reactor arrays. Lab on a Chip, 11(15), 2509–2517. https://doi.org/10.1039/c0lc00553c
Hatch,, A. C., Fisher,, J. S., Tovar,, A. R., Hsieh,, A. T., Lin,, R., Pentoney,, S. L., … Lee,, A. P. (2011). 1‐million droplet array with wide‐field fluorescence imaging for digital PCR. Lab on a Chip, 11(22), 3838–3845. https://doi.org/10.1039/c1lc20561g
Heredia,, N. J., Belgrader,, P., Wang,, S., Koehler,, R., Regan,, J., Cosman,, A. M., … Karlin‐Neumann,, G. (2013). Droplet digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods, 59(1), 183–186. https://doi.org/10.1016/j.ymeth.2012.09.012
Héritier,, S., Emile,, J. F., Barkaoui,, M. A., Thomas,, C., Fraitag,, S., Boudjemaa,, S., … Donadieu,, J. (2016). BRAF mutation correlates with high‐risk Langerhans cell Histiocytosis and increased resistance to first‐line therapy. Journal of Clinical Oncology, 34(25), 3023–3030. https://doi.org/10.1200/JCO.2015.65.9508
Hindson,, B. J., Ness,, K. D., Masquelier,, D. A., Belgrader,, P., Heredia,, N. J., Makarewicz,, A. J., … Colston,, B. W. (2011). High‐throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical Chemistry, 83(22), 8604–8610. https://doi.org/10.1021/ac202028g
Hsieh,, K., Zec,, H. C., Ma,, P. C., Rane,, T. D., & Wang,, T. H. (2015). Enhancing throughput of combinatorial droplet devices via droplet bifurcation, parallelized droplet fusion, and parallelized detection. Micromachines, 6(10), 1490–1504. https://doi.org/10.3390/mi6101434
Hu,, Y., Xu,, P., Luo,, J., He,, H., & Du,, W. (2017). Absolute quantification of H5‐subtype avian influenza viruses using droplet digital loop‐mediated isothermal amplification. Analytical Chemistry, 89(1), 745–750. https://doi.org/10.1021/acs.analchem.6b03328
Huang,, C. J., Fang,, W. F., Ke,, M. S., Chou,, H. Y. E., & Yang,, J. T. (2014). A biocompatible open‐surface droplet manipulation platform for detection of multi‐nucleotide polymorphism. Lab on a Chip, 14(12), 2057–2062. https://doi.org/10.1039/c4lc00089g
Huang,, J. T., Liu,, Y. J., Wang,, J., Xu,, Z. G., Yang,, Y., Shen,, F., … Liu,, S. M. (2015). Next generation digital PCR measurement of hepatitis B virus copy number in formalin‐fixed paraffin‐embedded hepatocellular carcinoma tissue. Clinical Chemistry, 61(1), 290–296. https://doi.org/10.1373/clinchem.2014.230227
Hung,, L. Y., Wu,, H. W., Hsieh,, K., & Lee,, G. B. (2014). Microfluidic platforms for discovery and detection of molecular biomarkers. Microfluidics and Nanofluidics, 16(5), 941–963. https://doi.org/10.1007/s10404-014-1354-6
Jambovane,, S., Kim,, D. J., Duin,, E. C., Kim,, S. K., & Hong,, J. W. (2011). Creation of stepwise concentration gradient in picoliter droplets for parallel reactions of matrix metalloproteinase II and IX. Analytical Chemistry, 83(9), 3358–3364. https://doi.org/10.1021/ac103217p
Jeannot,, E., Becette,, V., Campitelli,, M., Calmejane,, M. A., Lappartient,, E., Ruff,, E., … Sastre‐Garau,, X. (2016). Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus‐associated invasive carcinoma. The Journal of Pathology: Clinical Research, 2(4), 201–209. https://doi.org/10.1002/cjp2.47
Jing,, T., Lai,, Z., Wu,, L., Han,, J., Lim,, C. T., & Chen,, C.‐H. (2016). Single cell analysis of leukocyte protease activity using integrated continuous‐flow microfluidics. Analytical Chemistry, 88(23), 11750–11757. https://doi.org/10.1021/acs.analchem.6b03370
Jing,, T., Ramji,, R., Warkiani,, M. E., Han,, J., Lim,, C. T., & Chen,, C. H. (2015). Jetting microfluidics with size‐sorting capability for single‐cell protease detection. Biosensors and Bioelectronics, 66, 19–23. https://doi.org/10.1016/j.bios.2014.11.001
Joensson,, H. N., Samuels,, M. L., Brouzes,, E. R., Medkova,, M., Uhløn,, M., Link,, D. R., … Andersson‐Svahn,, H. (2009). Detection and analysis of low‐abundance cell‐surface biomarkers using enzymatic amplification in microfluidic droplets. Angewandte Chemie—International Edition, 48(14), 2518–2521. https://doi.org/10.1002/anie.200804326
Kang,, D.‐K., Ali,, M. M., Zhang,, K., Huang,, S. S., Peterson,, E., Digman,, M. A., … Zhao,, W. (2014). Rapid detection of single bacteria in unprocessed blood using integrated comprehensive droplet digital detection. Nature Communications, 5, 5427. https://doi.org/10.1038/ncomms6427
Kaushik,, A. M., Hsieh,, K., Chen,, L., Shin,, D. J., Liao,, J. C., & Wang,, T. H. (2017). Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform. Biosensors and Bioelectronics, 97, 260–266. https://doi.org/10.1016/j.bios.2017.06.006
Kelley,, K., Cosman,, A., Belgrader,, P., Chapman,, B., & Sullivan,, D. C. (2013). Detection of methicillin‐resistant Staphylococcus aureus by a duplex droplet digital PCR assay. Journal of Clinical Microbiology, 51(7), 2033–2039. https://doi.org/10.1128/JCM.00196-13
Kemna,, E. W. M., Segerink,, L. I., Wolbers,, F., Vermes,, I., & van den Berg,, A. (2013). Label‐free, high‐throughput, electrical detection of cells in droplets. The Analyst, 138(16), 4585–4592. https://doi.org/10.1039/c3an00569k
Khaw,, M. K., Ooi,, C. H., Mohd‐Yasin,, F., Vadivelu,, R., John,, J. S., & Nguyen,, N.‐T. (2016). Digital microfluidics with a magnetically actuated floating liquid marble. Lab on a Chip, 16(12), 2211–2218. https://doi.org/10.1039/C6LC00378H
Kim,, M., Pan,, M., Gai,, Y., Pang,, S., Han,, C., Yang,, C., & Tang,, S. K. Y. (2015). Optofluidic ultrahigh‐throughput detection of fluorescent drops. Lab on a Chip, 15(6), 1417–1423. https://doi.org/10.1039/C4LC01465K
Kim,, S. C., Premasekharan,, G., Clark,, I. C., Gemeda,, H. B., Paris,, P. L., & Abate,, A. R. (2017). Measurement of copy number variation in single cancer cells using rapid‐emulsification digital droplet MDA. Microsystems %26 Nanoengineering, 3, 17018. https://doi.org/10.1038/micronano.2017.18
Kinugasa,, H., Nouso,, K., Tanaka,, T., Miyahara,, K., Morimoto,, Y., Dohi,, C., … Yamamoto,, K. (2015). Droplet digital PCR measurement of HER2 in patients with gastric cancer. British Journal of Cancer, 112(10), 1652–1655. https://doi.org/10.1038/bjc.2015.129
Kiselinova,, M., Pasternak,, A. O., De Spiegelaere,, W., Vogelaers,, D., Berkhout,, B., & Vandekerckhove,, L. (2014). Comparison of droplet digital PCR and seminested real‐time PCR for quantification of cell‐associated HIV‐1 RNA. PLoS One, 9(1), e85999. https://doi.org/10.1371/journal.pone.0085999
Kiss,, M. M., Ortoleva‐Donnelly,, L., Reginald Beer,, N., Warner,, J., Bailey,, C. G., Colston,, B. W., … Leamon,, J. H. (2008). High‐throughput quantitative polymerase chain reaction in picoliter droplets. Analytical Chemistry, 80(23), 8975–8981. https://doi.org/10.1021/ac801276c
Köster,, S., Angilè,, F. E., Duan,, H., Agresti,, J. J., Wintner,, A., Schmitz,, C., … Weitz,, D. a. (2008). Drop‐based microfluidic devices for encapsulation of single cells. Lab on a Chip, 8(7), 1110–1115. https://doi.org/10.1039/b802941e
Laurent‐Puig,, P., Pekin,, D., Normand,, C., Kotsopoulos,, S. K., Nizard,, P., Perez‐Toralla,, K., … Taly,, V. (2015). Clinical relevance of KRAS‐mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti‐EGFR therapy. Clinical Cancer Research, 21(5), 1087–1097. https://doi.org/10.1158/1078-0432.CCR-14-0983
Leng,, S. X., McElhaney,, J. E., Walston,, J. D., Xie,, D., Fedarko,, N. S., & Kuchel,, G. A. (2008). ELISA and Multiplex Technologies for Cytokine Measurement in inflammation and aging research. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 63(8), 879–884. https://doi.org/10.1093/gerona/63.8.879
Li,, M., Diehl,, F., Dressman,, D., Vogelstein,, B., & Kinzler,, K. W. (2006). BEAMing up for detection and quantification of rare sequence variants. Nature Methods, 3(2), 95–97. https://doi.org/10.1038/NMETH850
Li,, Z., Leshansky,, A. M., Pismen,, L. M., & Tabeling,, P. (2015). Step‐emulsification in a microfluidic device. Lab on a Chip, 15(4), 1023–1031. https://doi.org/10.1039/C4LC01289E
Lim,, C. T., & Zhang,, Y. (2007). Bead‐based microfluidic immunoassays: The next generation. Biosensors and Bioelectronics, 22, 1197–1204. https://doi.org/10.1016/j.bios.2006.06.005
Lim,, J., Caen,, O., Vrignon,, J., Konrad,, M., Taly,, V., & Baret,, J. C. (2015). Parallelized ultra‐high throughput microfluidic emulsifier for multiplex kinetic assays. Biomicrofluidics, 9(3), 034101. https://doi.org/10.1063/1.4919415
Lim,, J., Vrignon,, J., Gruner,, P., Karamitros,, C. S., Konrad,, M., & Baret,, J.‐C. (2013). Ultra‐high throughput detection of single cellβ‐galactosidase activity in droplets using micro‐optical lens array. Applied Physics Letters, 103, 103. https://doi.org/10.1063/1.4830046
Liu,, X., Painter,, R. E., Enesa,, K., Holmes,, D., Whyte,, G., Garlisi,, C. G., … Smith,, C. A. (2016). High‐throughput screening of antibiotic‐resistant bacteria in picodroplets. Lab on a Chip, 16, 1636–1643. https://doi.org/10.1039/C6LC00180G
Luft,, L. M., Gill,, M. J., & Church,, D. L. (2011). HIV‐1 viral diversity and its implications for viral load testing: Review of current platforms. International Journal of Infectious Diseases, 15(10), e661–e670. https://doi.org/10.1016/j.ijid.2011.05.013
Luo,, J., Li,, J., Yang,, H., Yu,, J., & Wei,, H. (2017). Accurate detection of methicillin‐resistant Staphylococcus aureus in mixtures utilizing single bacterial duplex droplet digital PCR. Journal of Clinical Microbiology, (July), JCM.00716‐17. https://doi.org/10.1128/JCM.00716-17
Luther,, S. K., Will,, S., & Braeuer,, A. (2014). Phase‐specific Raman spectroscopy for fast segmented microfluidic flows. Lab on a Chip, 14, 2–5. https://doi.org/10.1039/c4lc00428k
Mach,, K. E., Wong,, P. K., & Liao,, J. C. (2011). Biosensor diagnosis of urinary tract infections: A path to better treatment? Trends in Pharmacological Sciences, 32, 330–336. https://doi.org/10.1016/j.tips.2011.03.001
Macosko,, E. Z., Basu,, A., Satija,, R., Nemesh,, J., Shekhar,, K., Goldman,, M., … McCarroll,, S. A. (2015). Highly parallel genome‐wide expression profiling of individual cells using nanoliter droplets. Cell, 161(5), 1202–1214. https://doi.org/10.1016/j.cell.2015.05.002
Mancini,, N., Carletti,, S., Ghidoli,, N., Cichero,, P., Burioni,, R., & Clementi,, M. (2010). The era of molecular and other non‐culture‐based methods in diagnosis of sepsis. Clinical Microbiology Reviews, 23, 235–251. https://doi.org/10.1128/CMR.00043-09
März,, A., Henkel,, T., Cialla,, D., Schmitt,, M., & Popp,, J. (2011). Droplet formation via flow‐through microdevices in Raman and surface enhanced Raman spectroscopy—Concepts and applications. Lab on a Chip, 11(21), 3584–3592. https://doi.org/10.1039/c1lc20638a
Matsubara,, Y., Kerman,, K., Kobayashi,, M., Yamamura,, S., Morita,, Y., & Tamiya,, E. (2005). Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes. Biosensors and Bioelectronics, 20, 1482–1490. https://doi.org/10.1016/j.bios.2004.07.002
Mazutis,, L., Araghi,, A. F., Miller,, O. J., Baret,, J. C., Frenz,, L., Janoshazi,, A., … Ryckelynck,, M. (2009). Droplet‐based microfluidic systems for high‐throughput single DNA molecule isothermal amplification and analysis. Analytical Chemistry, 81(12), 4813–4821. https://doi.org/10.1021/ac900403z
Mazutis,, L., Gilbert,, J., Ung,, W. L., Weitz,, D. A., Griffiths,, A. D., & Heyman,, J. A. (2013). Single‐cell analysis and sorting using droplet‐based microfluidics. Nature Protocols, 8(5), 870–891. https://doi.org/10.1038/nprot.2013.046
Micheel,, C. M., & Ball,, J. R. (2010). Evaluation of biomarkers and surrogate endpoints in chronic disease. Washington, DC: The National Academies Press. https://doi.org/10.17226/12869
Millar,, B. C., Xu,, J., & Moore,, J. E. (2007). Molecular diagnostics of medically important bacterial infections. Current Issues in Molecular Biology.
Miller,, E. M., & Wheeler,, A. R. (2008). A digital microfluidic approach to homogeneous enzyme assays. Analytical Chemistry, 80(5), 1614–1619. https://doi.org/10.1021/ac702269d
Mukaide,, M., Sugiyama,, M., Korenaga,, M., Murata,, K., Kanto,, T., Masaki,, N., & Mizokami,, M. (2014). High‐throughput and sensitive next‐generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70. Journal of Virological Methods, 207, 169–177. https://doi.org/10.1016/j.jviromet.2014.07.006
Nahavandi,, S., Baratchi,, S., Soffe,, R., Tang,, S.‐Y., Nahavandi,, S., Mitchell,, A., & Khoshmanesh,, K. (2014). Microfluidic platforms for biomarker analysis. Lab on a Chip, 14(9), 1496–1514. https://doi.org/10.1039/c3lc51124c
Najah,, M., Mayot,, E., Mahendra‐Wijaya,, I. P., Griffiths,, A. D., Ladame,, S., & Drevelle,, A. (2013). New glycosidase substrates for droplet‐based microfluidic screening. Analytical Chemistry, 85(20), 9807–9814. https://doi.org/10.1021/ac4022709
Nakashima,, T., Shimizu,, M., & Kukizaki,, M. (2000). Particle control of emulsion by membrane emulsification and its applications. Advanced Drug Delivery Reviews, 45(1), 47–56. https://doi.org/10.1016/S0169-409X(00)00099-5
Navin,, N. E. (2015). The first five years of single‐cell cancer genomics and beyond. Genome Research, 25, 1499–1507. https://doi.org/10.1101/gr.191098.115
Nelson,, W. C., & Kim,, C.‐J. (2012). Droplet actuation by Electrowetting‐on‐dielectric (EWOD): A review. Journal of Adhesion Science and Technology, 26(12–17), 1747–1771. https://doi.org/10.1163/156856111X599562
Ng,, E. X., Miller,, M. A., Jing,, T., & Chen,, C. H. (2016). Single cell multiplexed assay for proteolytic activity using droplet microfluidics. Biosensors and Bioelectronics, 81, 408–414. https://doi.org/10.1016/j.bios.2016.03.002
Ng,, E. X., Miller,, M. A., Jing,, T., Lauffenburger,, D. A., & Chen,, C.‐H. (2015). Low‐volume multiplexed proteolytic activity assay and inhibitor analysis through a pico‐injector array. Lab on a Chip, 15(4), 1153–1159. https://doi.org/10.1039/C4LC01162G
Nguyen,, N. T., Lassemono,, S., & Chollet,, F. A. (2006). Optical detection for droplet size control in microfluidic droplet‐based analysis systems. Sensors and Actuators, B: Chemical, 117(2), 431–436. https://doi.org/10.1016/j.snb.2005.12.010
Nisisako,, T., & Torii,, T. (2008). Microfluidic large‐scale integration on a chip for mass production of monodisperse droplets and particles. Lab on a Chip, 8(2), 287–293. https://doi.org/10.1039/B713141K
Niu,, X., & deMello,, A. J. (2012). Building droplet‐based microfluidic systems for biological analysis. Biochemical Society Transactions, 40(4), 615–623. https://doi.org/10.1042/bst20120005
Niu,, X., Zhang,, M., Peng,, S., Wen,, W., & Sheng,, P. (2007). Real‐time detection, control, and sorting of microfluidic droplets. Biomicrofluidics, 1(4), 044101. https://doi.org/10.1063/1.2795392
Novak,, R., Zeng,, Y., Shuga,, J., Venugopalan,, G., Fletcher,, D. A., Smith,, M. T., & Mathies,, R. A. (2011). Single‐cell multiplex gene detection and sequencing with microfluidically generated agarose emulsions. Angewandte Chemie—International Edition, 50(2), 390–395. https://doi.org/10.1002/anie.201006089
Ofner,, A., Moore,, D. G., Rühs,, P. A., Schwendimann,, P., Eggersdorfer,, M., Amstad,, E., … Studart,, A. R. (2016). High‐throughput step emulsification for the production of functional materials using a glass microfluidic device. Macromolecular Chemistry and Physics, 201600472, 1600472. https://doi.org/10.1002/macp.201600472
Otsuji,, K., Sasaki,, T., Tanaka,, A., Kunita,, A., Ikemura,, M., Matsusaka,, K., … Seto,, Y. (2016). Use of droplet digital PCR for quantitative and automatic analysis of the HER2 status in breast cancer patients. Breast Cancer Research and Treatment, 162(1), 11–18. https://doi.org/10.1007/s10549-016-4092-5
Oxnard,, G. R., Paweletz,, C. P., Kuang,, Y., Mach,, S. L., O`Connell,, A., Messineo,, M. M., … Jänne,, P. A. (2014). Noninvasive detection of response and resistance in EGFR‐mutant lung cancer using quantitative next‐generation genotyping of cell‐free plasma DNA. TL ‐ 20. Clinical Cancer Research, 20 VN‐r(6), 1698–1705. https://doi.org/10.1158/1078-0432.CCR-13-2482
Park,, S.‐Y., Teitell,, M. A., & Chiou,, E. P. Y. (2010). Single‐sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns. Lab on a Chip, 10(13), 1655–1661. https://doi.org/10.1039/c001324b
Park,, S.‐Y., Wu,, T.‐H., Chen,, Y., Teitell,, M. A., & Chiou,, P.‐Y. (2011). High‐speed droplet generation on demand driven by pulse laser‐induced cavitation. Lab on a Chip, 11(6), 1010–1012. https://doi.org/10.1039/c0lc00555j
Pavšič,, J., Žel,, J., & Milavec,, M. (2016). Digital PCR for direct quantification of viruses without DNA extraction. Analytical and Bioanalytical Chemistry, 408(1), 67–75. https://doi.org/10.1007/s00216-015-9109-0
Pekin,, D., Skhiri,, Y., Baret,, J.‐C., Le Corre,, D., Mazutis,, L., Salem,, C. B., … Taly,, V. (2011). Quantitative and sensitive detection of rare mutations using droplet‐based microfluidics. Lab on a Chip, 11(13), 2156–2166. https://doi.org/10.1039/C1LC20128J
Pekin,, D., & Taly,, V. (2017). Droplet‐based microfluidics digital PCR for the detection of KRAS mutations. Methods in Molecular Biology, 1547, 143–164. https://doi.org/10.1007/978-1-4939-6734-6_12
Pipper,, J., Zhang,, Y., Neuzil,, P., & Hsieh,, T. M. (2008). Clockwork PCR including sample preparation. Angewandte Chemie ‐ International Edition, 47(21), 3900–3904. https://doi.org/10.1002/anie.200705016
Postek,, W., Kaminski,, T., & Garstecki,, P. (2017). A passive microfluidic system based on step emulsification allows to generate libraries of nanoliter‐sized droplets from microliter droplets of varying and known concentration of sample. Lab on a Chip, 17, 1323–1331. https://doi.org/10.1039/C7LC00014F
Rački,, N., Morisset,, D., Gutierrez‐Aguirre,, I., & Ravnikar,, M. (2014). One‐step RT‐droplet digital PCR: A breakthrough in the quantification of waterborne RNA viruses. Analytical and Bioanalytical Chemistry, 406(3), 661–667. https://doi.org/10.1007/s00216-013-7476-y
Rane,, T. D., Chen,, L., Zec,, H. C., & Wang,, T. (2014). Microfluidic continuous flow digital loop‐mediated isothermal amplification (LAMP). Lab on a Chip, 15(3), 776–782. https://doi.org/10.1039/C4LC00901K
Rane,, T. D., Puleo,, C. M., Liu,, K. J., Zhang,, Y., Lee,, A. P., & Wang,, T. H. (2010). Counting single molecules in sub‐nanolitre droplets. Lab on a Chip, 10(2), 161–164. https://doi.org/10.1039/B917503B
Rane,, T. D., Zec,, H. C., Puleo,, C., Lee,, A. P., & Wang,, T.‐H. (2012). Droplet microfluidics for amplification‐free genetic detection of single cells. Lab on a Chip, 12(18), 3341–3347. https://doi.org/10.1039/c2lc40537g
Rane,, T. D., Zec,, H. C., & Wang,, T. H. (2015). A barcode‐free combinatorial screening platform for matrix metalloproteinase screening. Analytical Chemistry, 87(3), 1950–1956. https://doi.org/10.1021/ac504330x
Rane,, T. D., Zec,, H. C., & Wang,, T.‐H. (2012). A serial sample loading system: Interfacing multiwell plates with microfluidic devices. Journal of Laboratory Automation, 17(5), 370–377. https://doi.org/10.1177/2211068212455169
Reid,, A. L., Freeman,, J. B., Millward,, M., Ziman,, M., & Gray,, E. S. (2015). Detection of BRAF‐V600E and V600K in melanoma circulating tumour cells by droplet digital PCR. Clinical Biochemistry, 48(15), 999–1002. https://doi.org/10.1016/j.clinbiochem.2014.12.007
Rhee,, M., Light,, Y. K., Meagher,, R. J., & Singh,, A. K. (2016). Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples. PLoS One, 11(5), e0153699. https://doi.org/10.1371/journal.pone.0153699
Rinaldi,, A. (2011). Teaming up for biomarker future. EMBO Reports, 12(6), 500–504. https://doi.org/10.1038/embor.2011.90
Rondelez,, Y., Tresset,, G., Tabata,, K. V., Arata,, H., Fujita,, H., Takeuchi,, S., & Noji,, H. (2005). Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nature Biotechnology, 23(3), 361–365. https://doi.org/10.1038/nbt1072
Rosenfeld,, L., Lin,, T., Derda,, R., & Tang,, S. K. Y. (2014). Review and analysis of performance metrics of droplet microfluidics systems. Microfluidics and Nanofluidics, 16(5), 921–939. https://doi.org/10.1007/s10404-013-1310-x
Ruelle,, J., Yfantis,, V., Duquenne,, A., & Goubau,, P. (2014). Validation of an ultrasensitive digital droplet PCR assay for HIV‐2 plasma RNA quantification. Journal of the International AIDS Society, 17(4 Suppl 3), 19675. https://doi.org/10.7448/IAS.17.4.19675
Sacher,, A. G., Paweletz,, C., Dahlberg,, S. E., Alden,, R. S., O`Connell,, A., Feeney,, N., … Oxnard,, G. R. (2016). Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung Cancer. JAMA Oncology, 2(8), 1014–1022. https://doi.org/10.1001/jamaoncol.2016.0173
Sanmamed,, M., Fernández‐Landázuri,, S., Rodríguez,, C., Zárate,, R., Lozano,, M., Zubiri,, L., … González,, A. (2015). Quantitative cell‐free circulating BRAFV600E mutation analysis by use of droplet digital PCR in the follow‐up of patients with melanoma being treated with BRAF inhibitors. Clinical Chemistry, 61(1), 297–304. https://doi.org/10.1373/clinchem.2014.230235
Sawyers,, C. L. (2008). The cancer biomarker problem. Nature, 452(7187), 548–552. https://doi.org/10.1038/nature06913
Scheler,, O., Kaminski,, T. S., Ruszczak,, A., & Garstecki,, P. (2016). Dodecylresorufin (C12R) outperforms Resorufin in microdroplet bacterial assays. ACS Applied Materials and Interfaces, 8(18), 11318–11325. https://doi.org/10.1021/acsami.6b02360
Scheler,, O., Pacocha,, N., Debski,, P. R., Ruszczak,, A., Kaminski,, T. S., & Garstecki,, P. (2017). Optimized droplet digital CFU assay (ddCFU) provides precise quantification of bacteria over a dynamic range of 6 logs and beyond. Lab on a Chip, 17(11), 1980–1987. https://doi.org/10.1039/C7LC00206H
Schmid,, L., & Franke,, T. (2013). SAW‐controlled drop size for flow focusing. Lab on a Chip, 13(9), 1691–1694. https://doi.org/10.1039/c3lc41233d
Schmid,, L., & Franke,, T. (2014). Acoustic modulation of droplet size in a T‐junction. Applied Physics Letters, 104(13), 133501. https://doi.org/10.1063/1.4869536
Schuler,, F., Schwemmer,, F., Trotter,, M., Wadle,, S., Zengerle,, R., von Stetten,, F., & Paust,, N. (2015). Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA. Lab on a Chip, 15(13), 2759–2766. https://doi.org/10.1039/C5LC00291E
Shembekar,, N., Chaipan,, C., Utharala,, R., & Merten,, C. A. (2016). Droplet‐based microfluidics in drug discovery, transcriptomics and high‐throughput molecular genetics. Lab on a Chip, 16(8), 1314–1331. https://doi.org/10.1039/C6LC00249H
Shim,, J. U., Ranasinghe,, R. T., Smith,, C. A., Ibrahim,, S. M., Hollfelder,, F., Huck,, W. T. S., … Abell,, C. (2013). Ultrarapid generation of femtoliter microfluidic droplets for single‐molecule‐counting immunoassays. ACS Nano, 7(7), 5955–5964. https://doi.org/10.1021/nn401661d
Shin,, D. J., Zhang,, Y., & Wang,, T. H. (2014). A droplet microfluidic approach to single‐stream nucleic acid isolation and mutation detection. Microfluidics and Nanofluidics, 17(2), 425–430. https://doi.org/10.1007/s10404-013-1305-7
Stan,, C. A., Tang,, S. K. Y., & Whitesides,, G. M. (2009). Independent control of drop size and velocity in microfluidic flow‐focusing generators using variable temperature and flow rate. Analytical Chemistry, 81(6), 2399–2402. https://doi.org/10.1021/ac8026542
Strain,, M. C., Lada,, S. M., Luong,, T., Rought,, S. E., Gianella,, S., Terry,, V. H., … Richman,, D. D. (2013). Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One, 8(4), 2–9. https://doi.org/10.1371/journal.pone.0055943
Strimbu,, K., & Tavel,, J. A. (2011). What are biomarkers? Current Opininion in HIV and AIDS, 5(6), 463–466. https://doi.org/10.1097/COH.0b013e32833ed177.What
Sugiura,, S., Nakajima,, M., & Seki,, M. (2002). Effect of channel structure on microchannel emulsification. Langmuir, 18(15), 5708–5712. https://doi.org/10.1021/la025813a
Sykes,, P. J., Neoh,, S. H., Brisco,, M. J., Hughes,, E., Condon,, J., & Morley,, A. A. (1992). Quantitation of targets for PCR by use of limiting dilution. BioTechniques, 13(3), 444–449.
Syme,, C. D., Martino,, C., Yusvana,, R., Sirimuthu,, N. M. S., & Cooper,, J. M. (2012). Quantitative characterization of individual microdroplets using surface‐enhanced resonance raman scattering spectroscopy. Analytical Chemistry, 84(3), 1491–1495. https://doi.org/10.1021/ac202705a
Takahama,, T., Sakai,, K., Takeda,, M., Azuma,, K., Hida,, T., Hirabayashi,, M., … Nishio,, K. (2016). Detection of the T790M mutation of EGFR in plasma of advanced non–small cell lung cancer patients with acquired resistance to tyrosine kinase inhibitors (West Japan oncology group 8014LTR study) inhibitors (West Japan oncology group 8014LTR study). Oncotarget, 7(36), 8014–58499. https://doi.org/10.18632/oncotarget.11303
Taly,, V., Pekin,, D., Benhaim,, L., Kotsopoulos,, S. K., Le Corre,, D., Li,, X., … Laurent‐Puig,, P. (2013). Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clinical Chemistry, 59(12), 1722–1731. https://doi.org/10.1373/clinchem.2013.206359
Taly,, V., Pekin,, D., El Abed,, A., & Laurent‐Puig,, P. (2012). Detecting biomarkers with microdroplet technology. Trends in Molecular Medicine, 18, 405–416. https://doi.org/10.1016/j.molmed.2012.05.001
Tan,, S. H., Nguyen,, N. T., Yobas,, L., & Kang,, T. G. (2010). Formation and manipulation of ferrofluid droplets at a microfluidic T‐junction. Journal of Micromechanics and Microengineering, 20(4), 45004. https://doi.org/10.1088/0960-1317/20/4/045004
Tang,, M. Y. H., & Shum,, H. C. (2016). One‐step immunoassay of C‐reactive protein using droplet microfluidics. Lab on a Chip, 16(22), 4359–4365. https://doi.org/10.1039/C6LC01121G
Tanyeri,, M., Perron,, R., & Kennedy,, I. M. (2007). Lasing droplets in a microfabricated channel. Optics Letters, 32(17), 2529–2531. https://doi.org/10.1364/OL.32.002529
Taylor,, G. I. (1934). The formation of emulsions in definable fields of flow. Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 146(858), 501–523. https://doi.org/10.1098/rspa.1934.0169
Thorsen,, T., Roberts,, R. W., Arnold,, F. H., & Quake,, S. R. (2001). Dynamic pattern formation in a vesicle‐generating microfluidic device. Physical Review Letters, 86(18), 4163–4166. https://doi.org/10.1103/PhysRevLett.86.4163
Thress,, K. S., Brant,, R., Carr,, T. H., Dearden,, S., Jenkins,, S., Brown,, H., … Barrett,, J. C. (2015). EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross‐platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer, 90(3), 509–515. https://doi.org/10.1016/j.lungcan.2015.10.004
Tighe,, P. J., Ryder,, R. R., Todd,, I., & Fairclough,, L. C. (2015). ELISA in the multiplex era: Potentials and pitfalls. Proteomics—Clinical Applications., 9, 406–422. https://doi.org/10.1002/prca.201400130
Trypsteen,, W., Kiselinova,, M., Vandekerckhove,, L., & De Spiegelaere,, W. (2016). Diagnostic utility of droplet digital PCR for HIV reservoir quantification. Journal of Virus Eradication, 2(3), 162–169. Retrieved from. http://www.ncbi.nlm.nih.gov/pubmed/27482456\nhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4967968
Trypsteen,, W., Vynck,, M., De Neve,, J., Bonczkowski,, P., Kiselinova,, M., Malatinkova,, E., … De Spiegelaere,, W. (2015). ddpcRquant: Threshold determination for single channel droplet digital PCR experiments. Analytical and Bioanalytical Chemistry, 407(19), 5827–5834. https://doi.org/10.1007/s00216-015-8773-4
Unger,, M. A., Chou,, H., Thorsen,, T., Scherer,, A., & Quake,, S. R. (2000). Monolithic microfabricated valves and pumps by multilayer soft lithography. Science, 288(5463), 113–116. https://doi.org/10.1126/science.288.5463.113
van Ginkel,, J. H., Huibers,, M. M. H., van Es,, R. J. J., de Bree,, R., & Willems,, S. M. (2017). Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients. BMC Cancer, 17(1), 428. https://doi.org/10.1186/s12885-017-3424-0
Watanabe,, M., Kawaguchi,, T., Isa,, S. I., Ando,, M., Tamiya,, A., Kubo,, A., … Koh,, Y. (2015). Ultra‐sensitive detection of the pretreatment EGFR T790M mutation in non‐small cell lung cancer patients with an EGFR‐activating mutation using droplet digital PCR. Clinical Cancer Research, 21(15), 3552–3560. https://doi.org/10.1158/1078-0432.CCR-14-2151
Whitesides,, G. M. (2006). The origins and the future of microfluidics. Nature, 442(7101), 368–373. https://doi.org/10.1038/nature05058
Witte,, A. K., Fister,, S., Mester,, P., Schoder,, D., & Rossmanith,, P. (2016). Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR. Analytical and Bioanalytical Chemistry, 408(27), 7583–7593. https://doi.org/10.1007/s00216-016-9861-9
Wu,, Q., Jin,, W., Zhou,, C., Han,, S., Yang,, W., Zhu,, Q., … Mu,, Y. (2011). Integrated glass microdevice for nucleic acid purification, loop‐mediated isothermal amplification, and online detection. Analytical Chemistry, 83(9), 3336–3342. https://doi.org/10.1021/ac103129e
Xu,, J., & Attinger,, D. (2008). Drop on demand in a microfluidic chip. Journal of Micromechanics and Microengineering, 18(6), 65020. https://doi.org/10.1088/0960-1317/18/6/065020
Xu,, J. H., Li,, S. W., Tán,, J., Wang,, Y. J., & Luo,, G. S. (2006). Preparation of highly monodisperse droplet in a T‐junction microfluidic device. AICHE Journal, 52(9), 3005–3010. https://doi.org/10.1002/aic.10924
Xu,, P., Zheng,, X., Tao,, Y., & Du,, W. (2016). Cross‐Interface emulsification for generating size‐tunable droplets. Analytical Chemistry, 88(6), 3171–3177. https://doi.org/10.1021/acs.analchem.5b04510
Zagnoni,, M., Anderson,, J., & Cooper,, J. M. (2010). Hysteresis in multiphase microfluidics at a T‐junction. Langmuir, 26(12), 9416–9422. https://doi.org/10.1021/la1004243
Zec,, H., Rane,, T. D., & Wang,, T.‐H. (2012). Microfluidic platform for on‐demand generation of spatially indexed combinatorial droplets. Lab on a Chip, 12(17), 3055–3062. https://doi.org/10.1039/c2lc40399d
Zec,, H., Shin,, D. J., & Wang,, T.‐H. (2014). Novel droplet platforms for the detection of disease biomarkers. Expert Review of Molecular Diagnostics, 7159(February), 1–15. https://doi.org/10.1586/14737159.2014.945437
Zeng,, S., Li,, B., Su,, X., Qin,, J., & Lin,, B. (2009). Microvalve‐actuated precise control of individual droplets in microfluidic devices. Lab on a Chip, 9(10), 1340–1343. https://doi.org/10.1039/b821803j
Zhang,, P., Kaushik,, A., Hsieh,, K., & Wang,, T. H. (2017). Spatially encoded picoliter droplet groups for high‐throughput combinatorial analysis. In 2017 19th International Conference on Solid‐State Sensors, Actuators and Microsystems (TRANSDUCERS), Kaohsiung, Taiwan. https://doi.org/10.1109/TRANSDUCERS.2017.7994418
Zhang,, Y., & Nguyen,, N.‐T. (2017). Magnetic digital microfluidics—A review. Lab on a Chip, 17(6), 994–1008. https://doi.org/10.1039/C7LC00025A
Zhang,, Y., Park,, S., Liu,, K., Tsuan,, J., Yang,, S., & Wang,, T.‐H. (2011). A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification. Lab on a Chip, 11(3), 398–406. https://doi.org/10.1039/C0LC00296H
Zhang,, Y., Park,, S., Yang,, S., & Wang,, T. H. (2010). An all‐in‐one microfluidic device for parallel DNA extraction and gene analysis. Biomedical Microdevices, 12(6), 1043–1049. https://doi.org/10.1007/s10544-010-9458-6
Zhang,, Y., & Wang,, T. H. (2013). Full‐range magnetic manipulation of droplets via surface energy traps enables complex bioassays. Advanced Materials, 25(21), 2903–2908. https://doi.org/10.1002/adma.201300383
Zhong,, Q., Bhattacharya,, S., Kotsopoulos,, S., Olson,, J., Taly,, V., Griffiths,, A. D., … Larson,, J. W. (2011). Multiplex digital PCR: Breaking the one target per color barrier of quantitative PCR. Lab on a Chip, 11(13), 2167–2174. https://doi.org/10.1039/c1lc20126c
Zhu,, G., Ye,, X., Dong,, Z., Lu,, Y. C., Sun,, Y., Liu,, Y., … Liu,, X. (2015). Highly sensitive droplet digital PCR method for detection of EGFR‐activating mutations in plasma cell‐free DNA from patients with advanced non‐small cell lung cancer. Journal of Molecular Diagnostics, 17(3), 265–272. https://doi.org/10.1016/j.jmoldx.2015.01.004
Zhu,, P., & Wang,, L. (2017). Passive and active droplet generation with microfluidics: A review. Lab on a Chip, 17(1), 34–75. https://doi.org/10.1039/C6LC01018K
Zhu,, Y., & Fang,, Q. (2013). Analytical detection techniques for droplet microfluidics‐a review. Analytica Chimica Acta, 787, 24–35. https://doi.org/10.1016/j.aca.2013.04.064
Zonta,, E., Garlan,, F., Pécuchet,, N., Perez‐Toralla,, K., Caen,, O., Milbury,, C., … Taly,, V. (2016). Multiplex detection of rare mutations by Picoliter droplet based digital PCR: Sensitivity and specificity considerations. PLoS One, 11(7), e0159094. https://doi.org/10.1371/journal.pone.0159094

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

Droplet microfluidics for high‐sensitivity and high‐throughput detection and screening of disease biomarkers

Browse by Topic

Access to this WIREs title is by subscription only.

The latest WIREs articles in your inbox