Biotechnology and Bioprocess Engineering

, Volume 21, Issue 4, pp 573–579 | Cite as

Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors

  • Sung-Gil Hong
  • Jae Hyun Kim
  • Ryang Eun Kim
  • Seok-Joon Kwon
  • Dae Woo Kim
  • Hee-Tae Jung
  • Jonathan S. Dordick
  • Jungbae Kim
Research Paper

Abstract

Glucose oxidase (GOx) was immobilized onto graphene oxide (GRO) via three different preparation methods: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC), and enzyme adsorption, precipitation and crosslinking (EAPC). EAPC formulations, prepared via enzyme precipitation with 60% ammonium sulfate, showed 1,980 and 1,630 times higher activity per weight of GRO than those of EA and EAC formulations, respectively. After 59 days at room temperature, EAPC maintained 88% of initial activity, while EA and EAC retained 42 and 45% of their initial activities, respectively. These results indicate that the steps of precipitation and crosslinking in the EAPC formulation are critical to achieve high enzyme loading and stability of EAPC. EA, EAC and EAPC were used to prepare enzyme electrodes for use as glucose biosensors. Optimized EAPC electrode showed 93- and 25-fold higher sensitivity than EA and EAC, respectively. To further increase the sensitivity of EAPC electrode, multi-walled carbon nanotubes (MWCNTs) were mixed with EAPC for the preparation of enzyme electrode. Surprisingly, the EAPC electrode with additional 99.5 wt% MWCNTs showed 7,800-fold higher sensitivity than the EAPC electrode without MWCNT addition. Immobilization and stabilization of enzymes on GRO via the EAPC approach can be used for the development of highly sensitive biosensors as well as to achieve high enzyme loading and stability.

Keywords

enzyme adsorption/precipitation/crosslinking glucose oxidase graphene oxide biosensors carbon nanotubes 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Shao, Y. Y., J. Wang, H. Wu, J. Liu, I. A. Aksay, and Y. H. Lin (2010) Graphene based electrochemical sensors and biosensors: A review. Electroanal. 22: 1027–1036.CrossRefGoogle Scholar
  2. 2.
    Allen, M. J., V. C. Tung, and R. B. Kaner (2010) Honeycomb carbon: A review of graphene. Chem. Rev. 110: 132–145.CrossRefGoogle Scholar
  3. 3.
    Zhu, Y. W., S. Murali, W. W. Cai, X. S. Li, J. W. Suk, J. R. Potts, and R. S. Ruoff (2010) Graphene and graphene oxide: Synthesis, properties, and applications. Adv. Mater. 22: 3906–3924.CrossRefGoogle Scholar
  4. 4.
    Hass, J., W. A. d. Heer, and E. H. Conrad (2008) The growth and morphology of epitaxial multilayer graphene. J. Phys.: Condens. Matter 20: 1–27.Google Scholar
  5. 5.
    Stoller, M. D., S. Park, Y. Zhu, J. An, and R. S. Ruoff (2008) Graphene-based ultracapacitors. Nano Lett. 8: 3498–3502.CrossRefGoogle Scholar
  6. 6.
    Yoo, E., J. Kim, E. Hosono, H. -S. Zhou, T. Kudo, and I. Honma (2008) Large Reversible Li Storage of graphene nanosheet families for use in rechargeable lithium ion batteries. Nano Lett. 8: 2277–2282.CrossRefGoogle Scholar
  7. 7.
    Yoo, E., T. Okata, T. Akita, M. Kohyama, J. Nakamura, and I. Honma (2009) Enhanced electrocatalytic activity of Pt Subnanoclusters on graphene nanosheet surface. Nano Lett. 9: 2255–2259.CrossRefGoogle Scholar
  8. 8.
    Lu, C. H., H. H. Yang, C. L. Zhu, X. Chen, and G. N. Chen (2009) A graphene platform for sensing biomolecules. Angew. Chem. Int. Ed. 48: 4785–4787.CrossRefGoogle Scholar
  9. 9.
    Chen, H., M. B. Muller, K. J. Gilmore, G. G. Wallace, and D. Li (2008) Mechanically strong, electrically conductive, and biocompatible graphene paper. Adv. Mater. 20: 3557–3561.CrossRefGoogle Scholar
  10. 10.
    Liu, Z., J. T. Robinson, X. Sun, and H. Dai (2008) PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. J. Am. Chem. Soc. 130: 10876–10877.CrossRefGoogle Scholar
  11. 11.
    Carbone, M., L. Gorton, and R. Antiochia (2015) An overview of the latest graphene-based sensors for glucose detection: The effects of graphene defects. Electroanal. 27: 16–31.CrossRefGoogle Scholar
  12. 12.
    Liu, F., Y. Piao, K. S. Choi, and T. S. Seo (2012) Fabrication of free-standing graphene composite films as electrochemical biosensors. Carbon 50: 123–133.CrossRefGoogle Scholar
  13. 13.
    Choi, B. G., H. Park, T. J. Park, M. H. Yang, J. S. Kim, S. Y. Jang, N. S. Heo, S. Y. Lee, J. Kong, and W. H. Hong (2010) Solution chemistry of self-assembled graphene nanohybrids for high-performance flexible biosensors. Acs Nano 4: 2910–2918.CrossRefGoogle Scholar
  14. 14.
    Li, M. G., S. D. Xu, M. Tang, L. Liu, F. Gao, and Y. L. Wang (2011) Direct electrochemistry of horseradish peroxidase on graphene-modified electrode for electrocatalytic reduction towards H2O2. Electrochim. Acta 56: 1144–1149.CrossRefGoogle Scholar
  15. 15.
    Zhang, Q., S. J. Yang, J. Zhang, L. Zhang, P. L. Kang, J. H. Li, J. W. Xu, H. Zhou, and X. M. Song (2011) Fabrication of an electrochemical platform based on the self-assembly of graphene oxide-multiwall carbon nanotube nanocomposite and horseradish peroxidase: Direct electrochemistry and electrocatalysis. Nanotechnol. 22: 1–7.CrossRefGoogle Scholar
  16. 16.
    Qiu, J. D., J. Huang, and R. P. Liang (2011) Nanocomposite film based on graphene oxide for high performance flexible glucose biosensor. Sensor. Actuat. B-Chem. 160: 287–294.CrossRefGoogle Scholar
  17. 17.
    Unnikrishnan, B., S. Palanisamy, and S. -M. Chen (2013) A simple electrochemical approach to fabricate a glucose biosensor based on graphene–glucose oxidase biocomposite. Biosens. Bioelectron. 39: 70–75.CrossRefGoogle Scholar
  18. 18.
    Wang, Z. J., X. Z. Zhou, J. Zhang, F. Boey, and H. Zhang (2009) Direct electrochemical reduction of single-layer graphene oxide and subsequent functionalization with glucose oxidase. J. Phys. Chem. C 113: 14071–14075.CrossRefGoogle Scholar
  19. 19.
    Liu, Y., D. S. Yu, C. Zeng, Z. C. Miao, and L. M. Dai (2010) Biocompatible graphene oxide-based glucose biosensors. Langmuir 26: 6158–6160.CrossRefGoogle Scholar
  20. 20.
    Kim, R. E., S. G. Hong, S. Ha, and J. Kim (2014) Enzyme adsorption, precipitation and crosslinking of glucose oxidase and laccase on polyaniline nanofibers for highly stable enzymatic biofuel cells. Enz Microb. Technol. 66: 35–41.CrossRefGoogle Scholar
  21. 21.
    Kim, J. H., S. G. Hong, H. J. Sun, S. Ha, and J. Kim (2016) Precipitated and chemically-crosslinked laccase over polyaniline nanofiber for high performance phenol sensing. Chemosphere 143: 142–147.CrossRefGoogle Scholar
  22. 22.
    Hong, S. G., H. S. Kim, and J. Kim (2014) Highly stabilized lipase in polyaniline nanofibers for surfactant-mediated esterification of ibuprofen. Langmuir 30: 911–915.CrossRefGoogle Scholar
  23. 23.
    Kim, H., I. Lee, Y. Kwon, B. C. Kim, S. Ha, J. H. Lee, and J. Kim (2011) Immobilization of glucose oxidase into polyaniline nanofiber matrix for biofuel cell applications. Biosens. Bioelectron. 26: 3908–3913.CrossRefGoogle Scholar
  24. 24.
    Bergmeyer, H. U., Gawehn, K., Grassl, M. (1974) Methods of Enzymatic Analysis. Second edn. Academic Press Inc., NY, USA.Google Scholar
  25. 25.
    Mozhaev, V. V., M. V. Sergeeva, A. B. Belova, and Y. L. Khmelnitsky (1990) Multipoint attachment to a support protects enzyme from inactivation by organic-solvents -alpha-chymotrypsin in aqueous-solutions of alcohols and diols. Biotechnol. Bioeng. 35: 653–659.CrossRefGoogle Scholar
  26. 26.
    Mozhaev, V. V. (1993) Mechanism-based strategies for protein thermostabilization. Trends Biotechnol. 11: 88–95.CrossRefGoogle Scholar
  27. 27.
    Mozhaev, V. V., N. S. Melik-nubarov, M. V. Sergeeva, V. Šikšnis, and K. Martinek (1990) Strategy for stabilizing enzymes part one: Increasing stability of enzymes via their multi-point interaction with a support. Biocatal. 3: 179–187.CrossRefGoogle Scholar
  28. 28.
    Kim, J. H., S. -A. Jun, Y. Kwon, S. Ha, B. -I. Sang, and J. Kim (2015) Enhanced electrochemical sensitivity of enzyme precipitate coating (EPC)-based glucose oxidase biosensors with increased free CNT loadings. Bioelectrochem. 101: 114–119.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Sung-Gil Hong
    • 1
    • 2
  • Jae Hyun Kim
    • 1
    • 2
  • Ryang Eun Kim
    • 1
    • 2
  • Seok-Joon Kwon
    • 3
  • Dae Woo Kim
    • 4
  • Hee-Tae Jung
    • 4
  • Jonathan S. Dordick
    • 3
  • Jungbae Kim
    • 1
    • 2
  1. 1.Department of Chemical and Biological EngineeringKorea UniversitySeoulKorea
  2. 2.Green SchoolKorea UniversitySeoulKorea
  3. 3.Department of Chemical and Biological Engineering, and Center for Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  4. 4.Department of Chemical and Biomolecular Eng. (BK-21 plus) & KAIST Institute for Nano centuryKorea Advanced Institute of Science and TechnologyDaejeonKorea

Personalised recommendations