Advertisement

Droplets as Reaction Compartments for Protein Nanotechnology

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 996)

Abstract

Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets.

Key words

In vitro compartmentalization Directed evolution Protein engineering Microfluidics Water-in-oil emulsion 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported by the EU Marie-Curie network ProSA and the BBSRC. S.R.A.D. and M.F. are EU Marie-Curie fellows. F.H. holds an ERC Starting Investigator Grant.

References

  1. 1.
    Schaerli Y, Hollfelder F (2009) The potential of microfluidic water-in-oil droplets in experimental biology. Mol Biosyst 5:1392–1404PubMedCrossRefGoogle Scholar
  2. 2.
    Chen DL, Gerdts CJ, Ismagilov RF (2005) Using Microfluidics to Observe the Effect of Mixing on Nucleation of Protein Crystals. J Am Chem Soc 127:9672–9673PubMedCrossRefGoogle Scholar
  3. 3.
    Huebner A, Bratton D, Whyte G, Yang M, deMello AJ, Abell C, Hollfelder F (2009) Static microdroplet arrays: a microfluidic device for droplet trapping, incubation and release for enzymatic and cell-based assays. Lab Chip 9:692–698PubMedCrossRefGoogle Scholar
  4. 4.
    Huebner A, Srisa-Art M, Holt D, Abell C, Hollfelder F, Demello AJ, Edel JB (2007) Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Commun 2007:1218–1220CrossRefGoogle Scholar
  5. 5.
    Hufnagel H, Huebner A, Gulch C, Guse K, Abell C, Hollfelder F (2009) An integrated cell culture lab on a chip: modular microdevices for cultivation of mammalian cells and delivery into microfluidic microdroplets. Lab Chip 9:1576–1582PubMedCrossRefGoogle Scholar
  6. 6.
    Köster S, Angile FE, Duan H, Agresti JJ, Wintner A, Schmitz C, Rowat AC, Merten CA, Pisignano D, Griffiths AD, Weitz DA (2008) Drop-based microfluidic devices for encapsulation of single cells. Lab Chip 8:1110–1115PubMedCrossRefGoogle Scholar
  7. 7.
    Clausell-Tormos J, Lieber D, Baret J-C, El-Harrak A, Miller OJ, Frenz L, Blouwolff J, Humphry KJ, Köster S, Duan H, Holtze C, Weitz DA, Griffiths AD, Merten CA (2008) Droplet-Based Microfluidic Platforms for the Encapsulation and Screening of Mammalian Cells and Multicellular Organisms. Chem Biol 15:427–437PubMedCrossRefGoogle Scholar
  8. 8.
    Brouzes E, Medkova M, Savenelli N, Marran D, Twardowski M, Hutchison JB, Rothberg JM, Link DR, Perrimon N, Samuels ML (2009) Droplet microfluidic technology for single-cell high-throughput screening. Proc Natl Acad Sci USA 106:14195–14200PubMedCrossRefGoogle Scholar
  9. 9.
    Shim JU, Olguin LF, Whyte G, Scott D, Babtie A, Abell C, Huck WTS, Hollfelder F (2009) Simultaneous determination of gene expression and enzymatic activity in individual bacterial cells in microdroplet compartments. J Am Chem Soc 131:15251–15256PubMedCrossRefGoogle Scholar
  10. 10.
    El Debs BW, Tschulena U, Griffiths AD, Merten CA (2011) A competitive co-cultivation assay for cancer drug specificity evaluation. J Biomol Screen 16:818–824PubMedCrossRefGoogle Scholar
  11. 11.
    Shim J-u, Patil SN, Hodgkinson JT, Bowden SD, Spring DR, Welch M, Huck WTS, Hollfelder F, Abell C (2011) Controlling the contents of microdroplets by exploiting the permeability of PDMS. Lab Chip 11:1132–1137PubMedCrossRefGoogle Scholar
  12. 12.
    Huebner A, Olguin LF, Bratton D, Whyte G, Huck WTS, deMello AJ, Edel JB, Abell C, Hollfelder F (2008) Development of quantitative cell-based enzyme assays in microdroplets. Anal Chem 80:3890–3896PubMedCrossRefGoogle Scholar
  13. 13.
    Damean N, Olguin LF, Hollfelder F, Abell C, Huck WTS (2009) Simultaneous measurement of reactions in microdroplets filled by concentration gradients. Lab Chip 9:1707–1713PubMedCrossRefGoogle Scholar
  14. 14.
    Agresti JJ, Antipov E, Abate AR, Ahn K, Rowat AC, Baret JC, Marquez M, Klibanov AM, Griffiths AD, Weitz DA (2010) Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc Natl Acad Sci USA 107:4004–4009PubMedCrossRefGoogle Scholar
  15. 15.
    Aharoni A, Amitai G, Bernath K, Magdassi S, Tawfik DS (2005) High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem Biol 12:1281–1289PubMedCrossRefGoogle Scholar
  16. 16.
    Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD (2005) High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions. Chem Biol 12:1291–1300PubMedCrossRefGoogle Scholar
  17. 17.
    Cohen HM, Tawfik DS, Griffiths AD (2004) Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization. Prot Eng Des Sel 17:3–11CrossRefGoogle Scholar
  18. 18.
    Kintses B, Hein C, Mohamed MF, Fischlechner M, Courtois F, Lainé C, Hollfelder F (2012) Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem Biol 19:1001–1009Google Scholar
  19. 19.
    Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145PubMedCrossRefGoogle Scholar
  20. 20.
    Umbanhowar PB, Prasad V, Weitz DA (2000) Monodisperse Emulsion Generation via Drop Break Off in a Coflowing Stream. Langmuir 16:347–351CrossRefGoogle Scholar
  21. 21.
    Theberge AB, Courtois F, Schaerli Y, Fischlechner M, Abell C, Hollfelder F, Huck WTS (2010) Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology. Angew Chem Int Ed Engl 49:5846–5868PubMedGoogle Scholar
  22. 22.
    Kintses B, van Vliet LD, Devenish SR, Hollfelder F (2010) Microfluidic droplets: new integrated workflows for biological experiments. Curr Opin Chem Biol 14:548–555PubMedCrossRefGoogle Scholar
  23. 23.
    Holtze C, Rowat AC, Agresti JJ, Hutchison JB, Angilè FE, Schmitz CHJ, Köster S, Duan H, Humphry KJ, Scanga RA, Johnson JS, Pisignano D, Weitz DA (2008) Biocompatible surfactants for water-in-fluorocarbon emulsions. Lab Chip 8:1632PubMedCrossRefGoogle Scholar
  24. 24.
    Chen C-H, Sarkar A, Song Y-A, Miller MA, Kim SJ, Griffith LG, Lauffenburger DA, Han J (2011) Enhancing Protease Activity Assay in Droplet-Based Microfluidics Using a Biomolecule Concentrator. J Am Chem Soc 133:10368–10371PubMedCrossRefGoogle Scholar
  25. 25.
    Courtois F, Olguin LF, Whyte G, Theberge AB, Huck WTS, Hollfelder F, Abell C (2009) Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays. Anal Chem 81:3008–3016PubMedCrossRefGoogle Scholar
  26. 26.
    Stein V, Johnsson K, Hollfelder F (2007) A Covalent Chemical Genotype–Phenotype Linkage for in vitro Protein Evolution. Chembiochem 8:2191–2194PubMedCrossRefGoogle Scholar
  27. 27.
    Kaltenbach M, Stein V, Hollfelder F (2011) SNAP Dendrimers: Multivalent Protein Display on Dendrimer-Like DNA for Directed Evolution. Chembiochem 12:2208–2216PubMedCrossRefGoogle Scholar
  28. 28.
    Huebner A, Srisa-Art M, Holt D, Abell C, Hollfelder F, deMello AJ, Edel JB (2007) Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Commun (12):1218–1220CrossRefGoogle Scholar
  29. 29.
    Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656PubMedCrossRefGoogle Scholar
  30. 30.
    Courtois F, Olguin LF, Whyte G, Bratton D, Huck WTS, Abell C, Hollfelder F (2008) An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets. Chembiochem 9:439–446PubMedCrossRefGoogle Scholar
  31. 31.
    Agresti JJ, Kelly BT, Jäschke A, Griffiths AD (2005) Selection of ribozymes that catalyse multiple-turnover Diels–Alder cycloadditions by using in vitro compartmentalization. Proc Natl Acad Sci USA 102:16170–16175PubMedCrossRefGoogle Scholar
  32. 32.
    Griffiths AD, Tawfik DS (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J 22:24–35PubMedCrossRefGoogle Scholar
  33. 33.
    Stein V, Sielaff I, Johnsson K, Hollfelder F (2007) A covalent chemical genotype-phenotype linkage for in vitro protein evolution. Chembiochem 8:2191–2194PubMedCrossRefGoogle Scholar
  34. 34.
    Miller OJ, Bernath K, Agresti JJ, Amitai G, Kelly BT, Mastrobattista E, Taly V, Magdassi S, Tawfik DS, Griffiths AD (2006) Directed evolution by in vitro compartmentalization. Nat Meth 3:561–570CrossRefGoogle Scholar
  35. 35.
    Kaltenbach M, Devenish SRA, Hollfelder F (2012) A simple method to evaluate the biochemical compatibility of oil/surfactant mixtures for experiments in microdroplets. Lab Chip 12:4185–92Google Scholar
  36. 36.
    Schaerli Y, Wootton RC, Robinson T, Stein V, Dunsby C, Neil MA, French PM, deMello AJ, Abell C, Hollfelder F (2009) Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets. Anal Chem 81:302–306PubMedCrossRefGoogle Scholar
  37. 37.
    Frenz L, Blank K, Brouzes E, Griffiths AD (2009) Reliable microfluidic on-chip incubation of droplets in delay-lines. Lab Chip 9:1344–1348PubMedCrossRefGoogle Scholar
  38. 38.
    Bertschinger J, Grabulovski D, Neri D (2007) Selection of single domain binding proteins by covalent DNA display. Prot Eng Des Sel 20:57CrossRefGoogle Scholar
  39. 39.
    Bertschinger J, Neri D (2004) Covalent DNA display as a novel tool for directed evolution of proteins in vitro. Prot Eng Des Sel 17:699CrossRefGoogle Scholar
  40. 40.
    Doi N, Yanagawa H (1999) STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro. FEBS Lett 457:227–230PubMedCrossRefGoogle Scholar
  41. 41.
    Yonezawa M, Doi N, Higashinakagawa T, Yanagawa H (2004) DNA display of biologically active proteins for in vitro protein selection. J Biochem 135:285PubMedCrossRefGoogle Scholar
  42. 42.
    Yonezawa M, Doi N, Kawahashi Y, Higashinakagawa T, Yanagawa H (2003) DNA display for in vitro selection of diverse peptide libraries. Nuc Acids Res 31:e118CrossRefGoogle Scholar
  43. 43.
    Kaltenbach M, Hollfelder F (2011) SNAP display: In vitro protein evolution in microdroplets. In: Jackson RH, Douthwaite JA (eds) Ribosome Display and Related Technologies: Methods and Protocols. Humana Press (Springer Protocols), LondonGoogle Scholar
  44. 44.
    Kiss MM, Ortoleva-Donnelly L, Beer NR, Warner J, Bailey CG, Colston BW, Rothberg JM, Link DR, Leamon JH (2011) High-Throughput Quantitative Polymerase Chain Reaction in Picoliter Droplets. Anal Chem 80:8975–8981CrossRefGoogle Scholar
  45. 45.
    Ghadessy FJ, Ong JL, Holliger P (2001) Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA 98:4552–4557PubMedCrossRefGoogle Scholar
  46. 46.
    d’ Abbadie M, Hofreiter M, Vaisman A, Loakes D, Gasparutto D, Cadet J, Woodgate R, Pääbo S, Holliger P (2007) Molecular breeding of polymerases for amplification of ancient DNA. Nat Biotechnol 25:939–943CrossRefGoogle Scholar
  47. 47.
    Ghadessy FJ, Ramsay N, Boudsocq F, Loakes D, Brown A, Iwai S, Vaisman A, Woodgate R, Holliger P (2004) Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Nat Biotechnol 22:755–759PubMedCrossRefGoogle Scholar
  48. 48.
    Mazutis L, Araghi AF, Miller OJ, Baret JC, Frenz L, Janoshazi A, Taly V, Miller BJ, Hutchison JB, Link D, Griffiths AD, Ryckelynck M (2009) Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis. Anal Chem 81:4813–4821PubMedCrossRefGoogle Scholar
  49. 49.
    Paegel BM, Joyce GF (2010) Microfluidic Compartmentalized Directed Evolution. Chem Biol 17:717–724PubMedCrossRefGoogle Scholar
  50. 50.
    Jousse F, Farr R, Link DR, Fuerstman MJ, Garstecki P (2006) Bifurcation of droplet flows within capillaries. Phys Rev E 74:036311CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK

Personalised recommendations

Cite protocol

Buy options