Skip to main content
SpringerLink
Log in

Quantitative measurements of free and immobilized RgDAAO Michaelis-Menten constant using an electrochemical assay reveal the impact of covalent cross-linking on substrate specificity

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Challenges facing enzyme-based electrochemical sensors include substrate specificity, batch to batch reproducibility, and lack of quantitative metrics related to the effect of enzyme immobilization. We present a quick, simple, and general approach for measuring the effect of immobilization and cross-linking on enzyme activity and substrate specificity. The method can be generalized for electrochemical biosensors using an enzyme that releases hydrogen peroxide during its catalytic cycle. Using as proof of concept RgDAAO-based electrochemical biosensors, we found that the Michaelis-Menten constant (Km) decreases post immobilization, hinting at alterations in the enzyme kinetic properties and thus substrate specificity. We confirm the decrease in Km electrochemically by characterizing the substrate specificity of the immobilized RgDAAO using chronoamperometry. Our results demonstrate that enzyme immobilization affects enzyme substrate specificity and this must be carefully evaluated during biosensor development.

Graphical abstract

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Ehret F, Wu H, Alexander SC, Devaraj NK. Electrochemical control of rapid bioorthogonal tetrazine ligations for selective functionalization of microelectrodes. J Am Chem Soc. 2015;137:13.

    Article  CAS  Google Scholar 

  2. Schneider E, Clark DS. Cytochrome P450 (CYP) enzymes and the development of CYP biosensors. Biosens Bioelectron. 2013;39(1):1–13.

    Article  CAS  PubMed  Google Scholar 

  3. Tak-Shing Ching C, Chang J-W, Sun T-P, Shieh H-L, Tsai C-L, Huang H-W, et al. An amperometric biosensor array for precise determination of homocysteine. Sensors Actuators B. 2010;152:94–8.

    Article  CAS  Google Scholar 

  4. Li Z, Yu Y, Li Z, Wu T, Yin J. The art of signal transforming: electrodes and their smart applications in electrochemical sensing. Anal Methods. 2015;7:9732.

    Article  CAS  Google Scholar 

  5. Feng W, Ji P. Enzymes immobilized on carbon nanotubes. Biotechnol Adv. 2011;29(6):889–95.

    Article  CAS  PubMed  Google Scholar 

  6. Pundir CS, Lata S, Narwal V. Biosensors for determination of D and L- amino acids: a review. Biosens Bioelectron. 2018;117:373–84.

    Article  CAS  PubMed  Google Scholar 

  7. Vasylieva N, Barnych B, Meiller A, Maucler C, Pollegioni L, Lin J-S, et al. Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation. Biosens Bioelectron. 2011;26:3993–4000.

    Article  CAS  PubMed  Google Scholar 

  8. Andler SM, Goddard JM. Transforming food waste: how immobilized enzymes can valorize waste streams into revenue streams. npj Sci Food. 2018;2(1):19.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rehm FBH, Chen S, Rehm BHA. Bioengineering toward direct production of immobilized enzymes: a paradigm shift in biocatalyst design. Bioengineered. 2018;9(1):6–11.

    Article  PubMed  Google Scholar 

  10. Neira HD, Herr AE. Kinetic analysis of enzymes immobilized in porous film arrays. Anal Chem. 2017;89(19):10311–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bartlett PN, Cooper JM. A review of the immobilization of enzymes in electropolymerized films. J Electroanal Chem. 1993;362:1–12.

    Article  CAS  Google Scholar 

  12. D’souza SF. Immobilization and Stabilization of biomaterials for biosensor applications. Appl Biochem Biotechnol. 2001;96.

  13. Kim D, Herr AE. Protein immobilization techniques for microfluidic assays. Biomicrofluidics. 2013;7(4).

  14. Marangoni AG. Immobilized enzymes. In: Enzyme kinetics. Hoboken, NJ, USA: John Wiley & Sons, Inc.; p. 116–20.

  15. Anne A, Cambril E, Chovin A, Demaille C. Touching surface-attached molecules with a microelectrode: mapping the distribution of redox-labeled macromolecules by electrochemical-atomic force microscopy. Anal Chem. 2010;82(15):6353–62.

    Article  CAS  PubMed  Google Scholar 

  16. Jensen UB, Ferapontova EE, Sutherland DS. Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes. 2012.

  17. Bourdillon C, Demaille C, Moiroux J, Savéant J-M. Activation and diffusion in the kinetics of adsorption and molecular recognition on surfaces. Enzyme-amplified electrochemical approach to biorecognition dynamics illustrated by the binding of antibodies to immobilized antigens. P Curr Opin Biotechnol. 1998;70(7):2401–8.

    Google Scholar 

  18. Limoges B, Savéant J-M, Yazidi D. Quantitative analysis of catalysis and inhibition at horseradish peroxidase monolayers immobilized on an electrode surface. J Am Chem Soc. 2003;125(30):9192–203.

    Article  CAS  PubMed  Google Scholar 

  19. Limoges B, Marchal D, Mavré F, Savéant JM. Electrochemistry of immobilized redox enzymes: kinetic characteristics of NADH oxidation catalysis at diaphorase monolayers affinity immobilized on electrodes. J Am Chem Soc. 2006;128(6):2084–92.

    Article  CAS  PubMed  Google Scholar 

  20. Nguyen BH, Nguyen BT, Van Vu H, Van Nguyen C, Nguyen DT, Nguyen LT, et al. Development of label-free electrochemical lactose biosensor based on graphene/poly(1,5-diaminonaphthalene) film. Curr Appl Phys. 2016;16(2):135–40.

    Article  Google Scholar 

  21. Polcari D, Perry SC, Pollegioni L, Geissler M, Mauzeroll J. Localized detection of d -serine by using an enzymatic amperometric biosensor and scanning electrochemical microscopy. ChemElectroChem. 2017;4(4):920–6.

    Article  CAS  Google Scholar 

  22. Pribil MM, Cortés-Salazar F, Andreyev EA, Lesch A, Karyakina EE, Voronin OG, et al. Rapid optimization of a lactate biosensor design using soft probes scanning electrochemical microscopy. J Electroanal Chem. 2014;731:112–8.

    Article  CAS  Google Scholar 

  23. Soldà A, Valenti G, Marcaccio M, Giorgio M, Pelicci PG, Paolucci F, et al. Glucose and lactate miniaturized biosensors for SECM-based high-spatial resolution analysis: a comparative study. ACS Sensors. 2017.

  24. Campos-Beltrán D, Konradsson-Geuken Å, Quintero JE, Marshall L. Amperometric self-referencing ceramic based microelectrode arrays for D-serine detection. Biosensors. 2018;8(1):28–34.

    Article  CAS  Google Scholar 

  25. Sri Kaja B, Lumor S, Besong S, Taylor B, Ozbay G. Investigating enzyme activity of immobilized Candida rugosa lipase. J Food Qual. 2018;2018:1–9.

    Article  CAS  Google Scholar 

  26. Baghayeri M, Zare EN, Lakouraj MM, Biosensor H, Fe N. A simple hydrogen peroxide biosensor based on a novel electro-magnetic poly(p-phenylenediamine)@Fe 3 O 4 nanocomposite. Biosens Bioelectron. 2014;55:259–65.

    Article  CAS  PubMed  Google Scholar 

  27. Moussa S, Mauzeroll J. Review—microelectrodes: an overview of probe development and bioelectrochemistry applications from 2013 to 2018. J Electrochem Soc. 2019;166(6):G25–38.

    Article  CAS  Google Scholar 

  28. Ottone C, Romero O, Urrutia P, Bernal C, Illanes A, Wilson L. Enzyme biocatalysis and sustainability. In: Nanostructured catalysts for environmental applications. Springer International Publishing; 2021. p. 383–413.

  29. Abolpour Homaei A, Sariri R, Vianello F, Stevanato R. Enzyme immobilization: an update. J Chem Biol. 2013.

  30. Arabacı N, Karaytuğ T, Demirbas A, Ocsoy I, Katı A. Nanomaterials for enzyme immobilization. In: Green synthesis of nanomaterials for bioenergy applications. Wiley; 2020. p. 165–90.

  31. Bolivar JM, Nidetzky B. On the relationship between structure and catalytic effectiveness in solid surface-immobilized enzymes: advances in methodology and the quest for a single-molecule perspective. Vol. 1868, Biochimica et Biophysica Acta - Proteins and Proteomics. Elsevier B.V.; 2020. p. 140333.

  32. Bolivar JM, Schelch S, Mayr T, Nidetzky B. Mesoporous silica materials labeled for optical oxygen sensing and their application to development of a silica-supported oxidoreductase biocatalyst. ACS Catal. 2015;5(10):5984–93.

    Article  CAS  Google Scholar 

  33. Grasso G, Fragai M, Rizzarelli E, Spoto G, Yeo KJ. A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111). J Am Soc Mass Spectrom. 2007;18(5):961–9.

    Article  CAS  PubMed  Google Scholar 

  34. Grasso G, D’agata R, Rizzarelli E, Spoto G, D’andrea L, Pedone C, et al. Activity of anchored human matrix metalloproteinase-1 catalytic domain on Au (111) surfaces monitored by ESI-MS †. J MASS Spectrom J Mass Spectrom. 2005;40:1565–71.

    Article  CAS  PubMed  Google Scholar 

  35. Meridor D, Gedanken A. Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin. Enzym Microb Technol. 2014;67:67–76.

    Article  CAS  Google Scholar 

  36. Zain ZM, O’Neill RD, Lowry JP, Pierce KW, Tricklebank M, Dewa A, et al. Development of an implantable d-serine biosensor for in vivo monitoring using mammalian d-amino acid oxidase on a poly (o-phenylenediamine) and Nafion-modified platinum-iridium disk electrode. Biosens Bioelectron. 2010;25(6):1454–9.

    Article  CAS  PubMed  Google Scholar 

  37. Maucler C, Pernot P, Vasylieva N, Pollegioni L, Marinesco SS. In vivo D-serine hetero-exchange through alanine-serine-cysteine (ASC) transporters detected by microelectrode biosensors. ACS Chem Neurosci. 2013;4:51.

    Article  CAS  Google Scholar 

  38. Zain ZM, Ghani SA, O’Neill RD. Amperometric microbiosensor as an alternative tool for investigation of D-serine in brain. Amino Acids. 2012;43(5):1887–94.

    Article  CAS  Google Scholar 

  39. Papouin T, Haydon P. D-serine measurements in brain slices or other tissue explants. Bio-Protocol. 2018;8(2).

  40. Pollegioni L, Piubelli L, Sacchi S, Molla G. Review Physiological functions of D-amino acid oxidases: from yeast to humans.

  41. MacKay MAB, Kravtsenyuk M, Thomas R, Mitchell ND, Dursun SM, Baker GB. D-serine: potential therapeutic agent and/or biomarker in schizophrenia and depression? Vol. 10, Frontiers in Psychiatry. Frontiers Media S.A.; 2019.

  42. Sacchi S. d-Serine metabolism: new insights into the modulation of d-amino acid oxidase activity. 1551.

  43. Rosini E, D’Antona P, Pollegioni L. Biosensors for D-amino acids: detection methods and applications. Int J Mol Sci. 2020;21(13):4574.

    Article  CAS  PubMed Central  Google Scholar 

  44. Polcari D, Kwan A, Van Horn MR, Danis L, Pollegioni L, Ruthazer ES, et al. Disk-shaped amperometric enzymatic biosensor for in vivo detection of D-serine. Anal Chem. 2014;86(7):3501–7.

    Article  CAS  PubMed  Google Scholar 

  45. Moussa S, Van Horn M, Shah A, Pollegioni L, Thibodeaux C, Ruthazer E, et al. A miniaturized enzymatic biosensor for detection of sensory-evoked D-serine release in the brain. J Electrochem Soc. 2021.

  46. Spehar-Délèze A-M, Anastasova S, Vadgama P. Electropolymerised phenolic films as internal barriers for oxidase enzyme biosensors. Electroanalysis. 2014;26(6):1335–44.

    Article  CAS  Google Scholar 

  47. Bin HS, Wu GW, Deng HH, Liu AL, Lin XH, Xia XH, et al. Choline and acetylcholine detection based on peroxidase-like activity and protein antifouling property of platinum nanoparticles in bovine serum albumin scaffold. Biosens Bioelectron. 2014;62:331–6.

    Article  CAS  Google Scholar 

  48. Cappelletti P, Piubelli L, Murtas G, Caldinelli L, Valentino M, Molla G, et al. Structure–function relationships in human d-amino acid oxidase variants corresponding to known SNPs. Biochim Biophys Acta - Proteins Proteomics. 2015;1854(9):1150–9.

    Article  CAS  Google Scholar 

  49. Rosini E, Caldinelli L, Piubelli L. Assays of D-amino acid oxidase activity. Front Mol Biosci. 2018;4:102.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Caligiuri A, D’Arrigo P, Rosini E, Pedrocchi-Fantoni G, Tessaro D, Molla G, et al. Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids. J Mol Catal B Enzym. 2008;50(2–4):93–8.

    Article  CAS  Google Scholar 

  51. Danis L, Polcari D, Kwan A, Gateman SM, Mauzeroll J. Fabrication of carbon, gold, platinum, silver, and mercury ultramicroelectrodes with controlled geometry. Anal Chem. 2015;87(5):2565–9.

    Article  CAS  PubMed  Google Scholar 

  52. Kangas MJ, Burks RM, Atwater J, Lukowicz RM, Williams P, Holmes AE. Colorimetric sensor arrays for the detection and identification of chemical weapons and explosives. Vol. 47, Critical reviews in analytical chemistry. Taylor and Francis Ltd.; 2017. p. 138–53.

  53. Bard AJ, Faulkner LR. Electrochemical methods: fundamentals and applications. Mol Biol. 2015;8:129–33.

    Google Scholar 

  54. Lefrou C, Cornut R. Analytical expressions for quantitative scanning electrochemical microscopy (SECM). ChemPhysChem. 2010;11(3):547–56.

    Article  CAS  PubMed  Google Scholar 

  55. Biemans EALM, Verhoeven-Duif NM, Gerrits J, Claassen JAHR, Kuiperij HB, Verbeek MM. CSF d-serine concentrations are similar in Alzheimer’s disease, other dementias, and elderly controls. Neurobiol Aging. 2016;42:213–6.

    Article  CAS  PubMed  Google Scholar 

  56. Kumashiro S, Hashimoto A, Nishikawa T. Free d-serine in post-mortem brains and spinal cords of individuals with and without neuropsychiatric diseases. Brain Res. 1995;681(1–2):117–25.

    Article  CAS  PubMed  Google Scholar 

  57. Dos Santos JCS, Rueda N, Torres R, Barbosa O, Gonçalves LRB, Fernandez-Lafuente R. Evaluation of divinylsulfone activated agarose to immobilize lipases and to tune their catalytic properties. Process Biochem. 2015;50(6):918–27.

    Article  CAS  Google Scholar 

  58. Coscolín C, Beloqui A, Martínez-Martínez M, Bargiela R, Santiago G, Blanco RM, et al. Controlled manipulation of enzyme specificity through immobilization-induced flexibility constraints. Appl Catal A Gen. 2018;565:59–67.

    Article  CAS  Google Scholar 

  59. Barbosa O, Torres R, Ortiz C, Berenguer-Murcia Á, Rodrigues RC, Fernandez-Lafuente R. Heterofunctional supports in enzyme immobilization: From traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules. 2013;14:2433–62.

    Article  CAS  PubMed  Google Scholar 

  60. Shu FR, Wilson GS. Rotating ring-disk enzyme electrode for surface catalysis studies. Anal Chem. 1976;48(12):1679–86.

    Article  CAS  PubMed  Google Scholar 

  61. Gonzalez-Navarro FF, Stilianova-Stoytcheva M, Renteria-Gutierrez L, Belanche-Muñoz LA, Flores-Rios BL, Ibarra-Esquer JE. Glucose oxidase biosensor modeling and predictors optimization by machine learning methods. Sensors (Switzerland). 2016;16(11):16–22.

    Article  CAS  Google Scholar 

  62. Fortier G, Brassard E, Bélanger D. Optimization of a polypyrrole glucose oxidase biosensor. Biosens Bioelectron. 1990;5(6):473–90.

    Article  CAS  PubMed  Google Scholar 

  63. Li J, Xiao L-T, Liu X-M, Zeng G-M, Huang G-H, Shen G-L, et al. Amperometric biosensor with HRP immobilized on a sandwiched nano-Au / polymerized m-phenylenediamine film and ferrocene mediator. Anal Bioanal Chem. 2003;376(6):902–7.

    Article  CAS  PubMed  Google Scholar 

  64. Shoja Y, Rafati AA, Ghodsi J. Enzymatic biosensor based on entrapment of d -amino acid oxidase on gold nanofilm/MWCNTs nanocomposite modified glassy carbon electrode by sol-gel network: analytical applications for d -alanine in human serum. Enzym Microb Technol. 2017;100:20–7.

    Article  CAS  Google Scholar 

  65. Kornienko N, Ly KH, Robinson WE, Heidary N, Zhang JZ, Reisner E. Advancing techniques for investigating the enzyme–electrode interface. Acc Chem Res. 2019;acs.accounts.9b00087.

  66. Kueng A, Kranz C, Lugstein A, Bertagnolli E, Mizaikoff B. Integrated AFM–SECM in tapping mode: simultaneous topographical and electrochemical imaging of enzyme activity. Angew Chem Int Ed. 2003;42(28):3238–40.

    Article  CAS  Google Scholar 

  67. Baleizão C, Berberan-Santos MN. Enzyme kinetics with a twist. J Math Chem. 2011;49:1949–60.

    Article  CAS  Google Scholar 

  68. Cordeiro CA, De Vries MG, Cremers TIFH, Westerink BHC. The role of surface availability in membrane-induced selectivity for amperometric enzyme-based biosensors. Sensors Actuators B Chem. 2016;223:679–88.

    Article  CAS  Google Scholar 

  69. Weltin A, Kieninger J, Enderle B, Gellner A-K, Fritsch B, Urban GA. Polymer-based, flexible glutamate and lactate microsensors for in vivo applications. Biosens Bioelectron. 2014;61:192–9.

    Article  CAS  PubMed  Google Scholar 

  70. Lata S, Pundir CS. Fabrication of an amperometric D-amino acid biosensor based on nickel hexacyanoferrate polypyrrole hybrid film deposited on glassy carbon electrode..

  71. Kaki SS, Adlercreutz P. Quantitative analysis of enzymatic fractionation of multiple substrate mixtures. Biotechnol Bioeng. 2013;110:78–86.

    Article  CAS  PubMed  Google Scholar 

Download references

Data transparency

All data is available from the authors upon reasonable request.

Funding

S.M. and J.M. received financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) and MITACS. LP was supported by Fondo di Ateneo per la Ricerca.

Author information

Authors and Affiliations

  1. Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec, H3A 0B8, Canada

    Siba Moussa, Danny Chhin & Janine Mauzeroll

  2. Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi deII’Insubria, via J. H. Dunant 3, 21100, Varese, Italy

    Loredano Pollegioni

Authors
  1. Siba Moussa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Danny Chhin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Loredano Pollegioni
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Janine Mauzeroll
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The manuscript was written through the contributions of all authors.

Corresponding author

Correspondence to Janine Mauzeroll.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Published in the topical collection Electrochemistry for Neurochemical Analysis with guest editors Ashley E. Ross and Alexander G. Zestos.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

ESM 1

(DOCX 1.44 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moussa, S., Chhin, D., Pollegioni, L. et al. Quantitative measurements of free and immobilized RgDAAO Michaelis-Menten constant using an electrochemical assay reveal the impact of covalent cross-linking on substrate specificity. Anal Bioanal Chem 413, 6793–6802 (2021). https://doi.org/10.1007/s00216-021-03273-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-021-03273-z

Keywords

Search

Navigation