Immobilization of Enzymes on Magnetic Beads Through Affinity Interactions

  • Mihaela Badea
  • Akhtar Hayat
  • Jean-Louis Marty
Part of the Methods in Molecular Biology book series (MIMB, volume 2100)


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.

Key words

Affinity interactions Magnetic beads Tagged enzyme Faster assay kinetics Enzyme immobilization 


  1. 1.
    Solé S, Merkoçi A, Alegret S (2001) New materials for electrochemical sensing III. Beads Trends Anal Chem 20:102–110CrossRefGoogle Scholar
  2. 2.
    Plata MR, Contento AM, Rios A (2010) State-of-the-art of (bio)chemical sensor developments in analytical Spanish groups. Sensors 10:2511–2576CrossRefGoogle Scholar
  3. 3.
    Richardson J, Hawkins P, Luxton R (2001) The use of coated paramagnetic particles as a physical label in a magneto-immunoassay. Biosens Bioelectron 16:989–993CrossRefGoogle Scholar
  4. 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 11:1–16CrossRefGoogle Scholar
  5. 5.
    Zhang J, Cass AEG (2000) Electrochemical analysis of immobilised chemical and genetic biotinylated alkaline phosphatase. Anal Chim Acta 408:241–247CrossRefGoogle Scholar
  6. 6.
    Yao D, Vlessidis AG, Evmiridis NP (2002) Development of an interference-free chemiluminescence method for monitoring acetylcholine and choline based on immobilized enzymes. Anal Chim Acta 462:199–208CrossRefGoogle Scholar
  7. 7.
    Esseghaier C, Bergaoui Y, Tlili A, Abdelghani A (2008) Impedance spectroscopy on immobilized streptavidin horseradish peroxidase layer for biosensing. Sensors Actuat B 134:112–116CrossRefGoogle Scholar
  8. 8.
    Cosnier S, Lepellec A (1999) Poly(pyrrole-biotin): a new polymer for biomolecule grafting on electrode surfaces. Electrochim Acta 44:1833–1836CrossRefGoogle Scholar
  9. 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 71:3692–3697CrossRefGoogle Scholar
  10. 10.
    Cosnier S, Gondran C, Lepellec A, Senillou A (2001) Controlled fabrication of glucose and catechol microbiosensors via electropolymerized biotinylated polypyrrole films. Anal Lett 34:61–70CrossRefGoogle Scholar
  11. 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 3:727–732CrossRefGoogle Scholar
  12. 12.
    Barhoumi H, Maaref A, Martelet C, Jaffrezic- Renault N (2008) Urease immobilization on biotinylated polypyrrole coated ChemFEC devices for urea biosensor development. IRBM 29:192–201CrossRefGoogle Scholar
  13. 13.
    Gast F-U, Franke I, Meiss G, Pingoud A (2001) Immobilization of sugar-non-specifi c nucleases by utilizing the streptavidin-biotin interactions. J Biotechnol 87:131–141CrossRefGoogle Scholar
  14. 14.
    Bucur B, Andreescu S, Marty J (2004) Affinity methods to immobilize acetylcholinesterases for manufacturing biosensors. Anal Lett 37:1571–1588CrossRefGoogle Scholar
  15. 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 530:1–6CrossRefGoogle Scholar
  16. 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 20:217–225CrossRefGoogle Scholar
  17. 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 129:218–224CrossRefGoogle Scholar
  18. 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 52:200–205CrossRefGoogle Scholar
  19. 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 65:94–96CrossRefGoogle Scholar
  20. 20.
    Rambihar C, Kernan K (2010) Magnetic bead-based fluorometric detection of lection-glycoprotein interactions. Talanta 81:1676–1680CrossRefGoogle Scholar
  21. 21.
    Neumann NP, Lampen JO (1969) Glycoprotein structure of yeast invertase. Biochemistry 8:3552–3556CrossRefGoogle Scholar
  22. 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 453:191–200CrossRefGoogle Scholar
  23. 23.
    Campas M, Bucur B, Andreescu S, Marty JL (2004) Application of oriented immobilisation methods to enzyme sensors. Curr Top Biotechnol 1:95–107Google Scholar
  24. 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 34:529–540CrossRefGoogle Scholar
  25. 25.
    Andreescu S, Fournier D, Marty JL (2003) Development of highly sensitive sensor based on bioengineered acetylcholinesterase immobilized by affinity method. Anal Lett 36:1865–1885CrossRefGoogle Scholar
  26. 26.
    Haddour N, Cosnier S, Gondran C (2005) Electrogeneration of a poly(pyrrole)-NTA chelator fi lm for a reversible oriented immobilization of histidine-tagged proteins. J Am Chem Soc 127:5752–5753CrossRefGoogle Scholar
  27. 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 23:506–512CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Mihaela Badea
    • 1
  • Akhtar Hayat
    • 2
  • Jean-Louis Marty
    • 3
  1. 1.Faculty of Medicine, Transilvania University of BrasovBrasovRomania
  2. 2.Interdisciplinary Research Centre in Biomedical Materials (IRCBM)COMSATS University IslamabadLahorePakistan
  3. 3.BAE, Universite de Perpignan Via DomitiaCeretFrance

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