Skip to main content
SpringerLink
Log in

Immobilization of Enzymes on Magnetic Beads Through Affinity Interactions

  • Protocol
  • First Online:
Immobilization of Enzymes and Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1051))

Abstract

The development of enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native enzyme could be used to create strong affinity bonds between the protein structure and a surface functionalized with the complementary affinity ligand. These immobilization techniques are based on affinity interactions between biotin and (strept)avidin molecules, lectins and sugars, or metal chelate and histidine tag.

Recent developments involve immobilization of tagged enzymes onto magnetic nanoparticles. These supports can improve the performance of immobilized biomolecules in analytical assay because magnetic beads provide a relative large numbers of binding sites for biochemical reactions resulting in faster assay kinetics.

This chapter describes immobilization procedures of tagged enzymes onto various magnetic beads.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Solé S, Merkoçi A, Alegret S (2001) New materials for electrochemical sensing III. Beads. Trends Anal Chem 20:102–110

    Article  Google Scholar 

  2. Plata MR, Contento AM, Rios A (2010) State-of-the-art of (bio)chemical sensor developments in analytical spanish groups. Sensors (Basel) 10:2511–2576

    Article  CAS  Google Scholar 

  3. Richardson J, Hawkins P, Luxton R (2001) The use of coated paramagnetic particles as a physical label in a magneto-immunoassay. Biosens Bioelectron 16:989–993

    Article  PubMed  CAS  Google Scholar 

  4. Nilsson J, Stahl S, Lundeberg J, Uhlen M, Nygren P-A (1997) Affinity fusion strategies for detection, purification and immobilization of recombinant proteins. Protein Expr Purif 11:1–16

    Article  PubMed  CAS  Google Scholar 

  5. Zhang J, Cass AEG (2000) Electrochemical analysis of immobilised chemical and genetic biotinylated alkaline phosphatase. Anal Chim Acta 408:241–247

    Article  CAS  Google Scholar 

  6. Yao D, Vlessidis AG, Evmiridis NP (2002) Development of an interference-free chemiluminescence method for monitoring aceylcholine and choline based on immobilized enzymes. Anal Chim Acta 462:199–208

    Article  CAS  Google Scholar 

  7. Esseghaier C, Bergaoui Y, Tlili A, Abdelghani A (2008) Impedance spectroscopy on immobilized streptavidin horseradish peroxidase layer for biosensing. Sensors Actuat B 134:112–116

    Article  CAS  Google Scholar 

  8. Cosnier S, Lepellec A (1999) Poly(pyrrole-biotin): a new polymer for biomolecule grafting on electrode surfaces. Electrochim Acta 44:1833–1836

    Article  CAS  Google Scholar 

  9. Cosnier S, Stoytcheva M, Senillou A, Perrot H, Furriel RPM, Leone FA (1999) A biotinylated conducting polypyrrole for the spatially controlled construction of an amperometric biosensor. Anal Chem 71:3692–3697

    Article  PubMed  CAS  Google Scholar 

  10. Cosnier S, Gondran C, Lepellec A, Senillou A (2001) Controlled fabrication of glucose and catechol microbiosensors via electropolymerized biotinylated polypyrrole films. Anal lett 34:61–70

    Article  CAS  Google Scholar 

  11. Mousty C, Lepellec A, Cosnier S, Novoa A, Marks RS (2001) Fabrication of organic phase biosensors based on multilayered polyphenol oxidase protected by an alginate coating. Electrochem Commun 3:727–732

    Article  CAS  Google Scholar 

  12. Barhoumi H, Maaref A, Martelet C, Jaffrezic-Renault N (2008) Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development. IRBM 29:192–201

    Article  Google Scholar 

  13. Gast F-U, Franke I, Meiss G, Pingoud A (2001) Immobilization of sugar-non-specific nucleases by utilizing the streptavidin-biotin interactions. J Biotechnol 87:131–141

    Article  PubMed  CAS  Google Scholar 

  14. Bucur B, Andreescu S, Marty J (2004) Affinity methods to immobilize acetylcholinesterases for manufacturing biosensors. Anal Lett 37:1571–1588

    Article  CAS  Google Scholar 

  15. Bucur B, Danet AF, Marty JL (2005) Cholinesterase immobilisation on the surface of screen-printed electrodes based on concanavalin A affinity. Anal Chim Acta 530:1–6

    Article  CAS  Google Scholar 

  16. Bucur B, Danet AF, Marty JL (2004) Versatile method of cholinesterase immobilisation via affinity bonds using concanavalin A applied to the construction of a scrren-printes biosensor. Biosens Bioelectron 20:217–225

    Article  PubMed  CAS  Google Scholar 

  17. Liu L, Chen Z, Yang S, Jin X, Lin X (2008) A novel inhibition biosensor constructed by layer-by-layer technique based on biospecific affinity for the determination of sulfide. Sensors Actuat B 129:218–224

    Article  CAS  Google Scholar 

  18. Yang S, Chen Z, Jin X, Lin X (2006) HRP biosensor based on sugar-lectin biospecific interactions for the determination of phenolic compounds. Electrochim Acta 52:200–205

    Article  CAS  Google Scholar 

  19. Anzai J-I, Kobayashi Y, Nakamura N, Hoshi T (2000) Use of Con A and mannose-labeled enzymes for the preparation of enzyme films for biosensors. Sensors Actuat B 65:94–96

    Article  CAS  Google Scholar 

  20. Rambihar C, Kernan K (2010) Magnetic bead-based fluorometric detection of lection-glycoprotein interactions. Talanta 81:1676–1680

    Article  PubMed  CAS  Google Scholar 

  21. Neumann NP, Lampen JO (1969) Glycoprotein structure of yeast invertase. Biochemistry 8:3552–3556

    Article  PubMed  CAS  Google Scholar 

  22. Halliwell CM, Simon E, Toh CS, Bartlett PN, Cass AEG (2002) Immobilisation of lactate dehydrogenase on poly(aniline)-poly(acrylate) and poly(aniline)-poly-(vinyl sulphonate) films for use in a lactate biosensor. Anal Chim Acta 453:191–200

    Article  CAS  Google Scholar 

  23. Campas M, Bucur B, Andreescu S, Marty JL (2004) Application of oriented immobilisation methods to enzyme sensors. Curr Top Biotechnol 1:95–107

    Google Scholar 

  24. Andreescu S, Magearu V, Lougarre A, Fournier D, Marty JL (2001) Immobilization of enzymes on screen-printed sensors via an histidine tail. Application to the detection of pesticides using modified cholinesterase. Anal Lett 34:529–540

    Article  CAS  Google Scholar 

  25. Andreescu S, Fournier D, Marty JL (2003) Development of highly sensitive sensor based on bioengineered acetylcholinesterase immobilized by affinity method. Anal Lett 36:1865–1885

    Article  CAS  Google Scholar 

  26. Haddour N, Cosnier S, Gondran C (2005) Electrogeneration of a poly(pyrrole)-NTA chelator film for a reversible oriented immobilization of histidine-tagged proteins. J ACS 127:5752–5753

    CAS  Google Scholar 

  27. Istamboulie G, Andreescu S, Marty JL, Noguer T (2007) Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase. Biosens Bioelectron 23:506–512

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. IMAGES EA, University of Perpignan, Perpignan Cedex, France

    Audrey Sassolas

  2. IMAGES EA 4218, University of Perpignan, Perpignan Cedex, France

    Akhtar Hayat & Jean-Louis Marty

Authors
  1. Audrey Sassolas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akhtar Hayat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Louis Marty
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Instituto de Catlisis y Petroleoquimica, CSIC, Madrid, Spain

    Jose M. Guisan

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, New York

About this protocol

Cite this protocol

Sassolas, A., Hayat, A., Marty, JL. (2013). Immobilization of Enzymes on Magnetic Beads Through Affinity Interactions. In: Guisan, J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology, vol 1051. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-550-7_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-550-7_10

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-549-1

  • Online ISBN: 978-1-62703-550-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation