Skip to main content
SpringerLink
Log in
SpringerLink
Log in

Droplet-Based Microfluidics Methods for Detecting Enzyme Inhibitors

  • Protocol
  • First Online:
Targeting Enzymes for Pharmaceutical Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2089))

Abstract

Sub-nanoliter droplets produced in microfluidic devices have gained an enormous importance for performing all kinds of biochemical assays. One of the main reasons is that the amounts of reagents employed can be reduced in approximately five orders of magnitude compared to conventional microplate assays. In this chapter, we describe how to carry out the design, fabrication, and operation of a microfluidic device that allows performing enzyme kinetics and enzyme inhibition assays in droplets. This procedure can be used effectively to screen a small size library of compounds. Then, we describe how to use this droplet microfluidic setup to screen for potential inhibitor compounds eluted from a coupled high-performance liquid chromatography (HPLC) system that separates crude natural extracts.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shang L, Cheng Y, Zhao Y (2017) Emerging droplet microfluidics. Chem Rev 117:7964–8040. https://doi.org/10.1021/acs.chemrev.6b00848

    Article  CAS  PubMed  Google Scholar 

  2. Nie Z, Seo MS, Xu S et al (2008) Emulsification in a microfluidic flow-focusing device: effect of the viscosities of the liquids. Microfluid Nanofluid 5:585–594. https://doi.org/10.1007/s10404-008-0271-y

    Article  CAS  Google Scholar 

  3. Umbanhowar PB, Prasad V, Weitz DA (2000) Monodisperse emulsion generation via drop break off in a coflowing stream. Langmuir 16:347–351. https://doi.org/10.1021/la990101e

    Article  CAS  Google Scholar 

  4. Shim JU, Ranasinghe RT, Smith CA et al (2013) Ultrarapid generation of femtoliter microfluidic droplets for single-molecule-counting immunoassays. ACS Nano 7:5955–5964. https://doi.org/10.1021/nn401661d

    Article  CAS  PubMed  Google Scholar 

  5. Price AK, Paegel BM (2016) Discovery in droplets. Anal Chem 88:339–353. https://doi.org/10.1021/acs.analchem.5b04139

    Article  CAS  PubMed  Google Scholar 

  6. Damean N, Olguin LF, Hollfelder F et al (2009) Simultaneous measurement of reactions in microdroplets filled by concentration gradients. Lab Chip 9:1707–1713. https://doi.org/10.1039/b821021g

    Article  CAS  PubMed  Google Scholar 

  7. Gielen F, van Vliet L, Koprowski BT et al (2013) A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition. Anal Chem 85:4761–4769. https://doi.org/10.1021/ac400480z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sjostrom SL, Joensson HN, Svahn HA (2013) Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors. Lab Chip 13:1754–1761. https://doi.org/10.1039/c3lc41398e

    Article  CAS  PubMed  Google Scholar 

  9. Hess D, Rane A, Demello AJ, Stavrakis S (2015) High-throughput, quantitative enzyme kinetic analysis in microdroplets using stroboscopic epifluorescence imaging. Anal Chem 87:4965–4972. https://doi.org/10.1021/acs.analchem.5b00766

    Article  CAS  PubMed  Google Scholar 

  10. Zhu Y, Fang Q (2013) Analytical detection techniques for droplet microfluidics—a review. Anal Chim Acta 787:24–35. https://doi.org/10.1016/j.aca.2013.04.064

    Article  CAS  PubMed  Google Scholar 

  11. Han Z, Li W, Huang Y, Zheng B (2009) Measuring rapid enzymatic kinetics by electrochemical method in droplet-based microfluidic devices with pneumatic valves. Anal Chem 81:5840–5845. https://doi.org/10.1021/ac900811y

    Article  CAS  PubMed  Google Scholar 

  12. Sun S, Kennedy RRT (2014) Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors. Anal Chem 86:9309–9314. https://doi.org/10.1021/ac502542z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vazquez B, Qureshi N, Oropeza-Ramos L, Olguin LF (2014) Effect of velocity on microdroplet fluorescence quantified by laser-induced fluorescence. Lab Chip 14:3550–3555. https://doi.org/10.1039/c4lc00654b

    Article  CAS  PubMed  Google Scholar 

  14. Dressler OJ, Casadevall i, Solvas X, deMello AJ (2017) Chemical and biological dynamics using droplet-based microfluidics. Annu Rev Anal Chem 10:1–24. https://doi.org/10.1146/annurev-anchem-061516-045219

    Article  Google Scholar 

  15. Mao Z, Guo F, Xie Y et al (2015) Label-free measurements of reaction kinetics using a droplet-based optofluidic device. J Lab Autom 20:17–24. https://doi.org/10.1177/2211068214549625

    Article  CAS  PubMed  Google Scholar 

  16. Baccouche A, Okumura S, Sieskind R et al (2017) Massively parallel and multiparameter titration of biochemical assays with droplet microfluidics. Nat Protoc 12:1912–1932. https://doi.org/10.1038/nprot.2017.092

    Article  CAS  PubMed  Google Scholar 

  17. Cai L-F, Zhu Y, Du G-S, Fang Q (2012) Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay. Anal Chem 84:446–452. https://doi.org/10.1021/ac2029198

    Article  CAS  PubMed  Google Scholar 

  18. Miller OJ, El A, Mangeat T et al (2012) High-resolution dose–response screening using droplet-based microfluidics. Proc Natl Acad Sci 109:378–383. https://doi.org/10.1073/pnas.1113324109

    Article  PubMed  Google Scholar 

  19. Gu S, Lu Y, Ding Y et al (2013) Droplet-based microfluidics for dose-response assay of enzyme inhibitors by electrochemical method. Anal Chim Acta 796:68–74. https://doi.org/10.1016/j.aca.2013.08.016

    Article  CAS  PubMed  Google Scholar 

  20. Wang X-L, Zhu Y, Fang Q (2014) Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay. Analyst 139:191–197. https://doi.org/10.1039/c3an01917a

    Article  CAS  PubMed  Google Scholar 

  21. Price AK, MacConnell AB, Paegel BM (2016) HνSABR: photochemical dose-response bead screening in droplets. Anal Chem 88:2904–2911. https://doi.org/10.1021/acs.analchem.5b04811

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Theberge AB, Whyte G, Huck WTS (2010) Generation of picoliter droplets with defined contents and concentration gradients from the separation of chemical mixtures. Anal Chem 82:3449–3453. https://doi.org/10.1021/ac1005316

    Article  CAS  PubMed  Google Scholar 

  23. Keyon ASA, Guijt RM, Bolch CJ, Breadmore MC (2014) Droplet microfluidics for postcolumn reactions in capillary electrophoresis. Anal Chem 86:11811–11818. https://doi.org/10.1021/ac5033963

    Article  CAS  PubMed  Google Scholar 

  24. Ochoa A, Álvarez-Bohórquez E, Castillero E, Olguin LF (2017) Detection of enzyme inhibitors in crude natural extracts using droplet-based microfluidics coupled to HPLC. Anal Chem 89:4889–4896. https://doi.org/10.1021/acs.analchem.6b04988

    Article  CAS  PubMed  Google Scholar 

  25. Holtze C, Rowat AC, Agresti JJ et al (2008) Biocompatible surfactants for water-in-fluorocarbon emulsions. Lab Chip 8:1632–1639. https://doi.org/10.1039/b806706f

    Article  CAS  PubMed  Google Scholar 

  26. Zhu S, Edmonds WF, Hillmyer MA, Lodge TP (2005) Synthesis and self-assembly of highly incompatible polybutadiene- poly(hexafluoropropylene oxide) diblock copolymers. J Polym Sci B Polym Phys 43:3685–3694. https://doi.org/10.1002/polb.20621

    Article  CAS  Google Scholar 

  27. Fischlechner M, Schaerli Y, Mohamed MF et al (2014) Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat Chem 6:791–796. https://doi.org/10.1038/nchem.1996

    Article  CAS  PubMed  Google Scholar 

  28. Hirata K, Ichii T, Suzuki H et al (2012) Fractal-shaped microchannel design for a kinetic analysis of biochemical reaction in a delay line. Microfluid Nanofluid 13:273–278. https://doi.org/10.1007/s10404-012-0958-y

    Article  CAS  Google Scholar 

  29. Haubert K, Drier T, Beebe D (2006) PDMS bonding by means of a portable, low-cost corona system. Lab Chip 6:1548–1549. https://doi.org/10.1039/b610567j

    Article  CAS  PubMed  Google Scholar 

  30. Ferraro D, Serra M, Filippi D et al (2019) Controlling the distance of highly confined droplets in a capillary by interfacial tension for merging on-demand. Lab Chip 9:136–146. https://doi.org/10.1039/C8LC01182F

    Article  Google Scholar 

  31. Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery. Wiley, Hoboken

    Google Scholar 

  32. Skhiri Y, Gruner P, Semin B et al (2012) Dynamics of molecular transport by surfactants in emulsions. Soft Matter 8:10618–10627. https://doi.org/10.1039/c2sm25934f

    Article  CAS  Google Scholar 

  33. Exnowitz F, Meyer B, Hackl T (2012) NMR for direct determination of Km and Vmax of enzyme reactions based on the Lambert W function-analysis of progress curves. Biochim Biophys Acta Proteins Proteomics 1824:443–449. https://doi.org/10.1016/j.bbapap.2011.10.011

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank CONACyT (284240) and PAIP-UNAM (5000-9023) for financial support.

Author information

Authors and Affiliations

  1. Facultad de Química, Laboratorio de Biofísicoquímica, Universidad Nacional Autónoma de México (UNAM), Ciudad de México, Mexico

    Abraham Ochoa, Frida Trejo & Luis F. Olguín

Authors
  1. Abraham Ochoa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frida Trejo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luis F. Olguín
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luis F. Olguín .

Editor information

Editors and Affiliations

  1. Department of Biotechnology, Agricultural University of Athens, Athens, Greece

    Nikolaos E. Labrou

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ochoa, A., Trejo, F., Olguín, L.F. (2020). Droplet-Based Microfluidics Methods for Detecting Enzyme Inhibitors. In: Labrou, N. (eds) Targeting Enzymes for Pharmaceutical Development. Methods in Molecular Biology, vol 2089. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0163-1_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0163-1_14

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0162-4

  • Online ISBN: 978-1-0716-0163-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation