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Immobilization of Horseradish Peroxidase Enzyme on Nanoporous Titanium Dioxide Electrodes and Its Structural and Electrochemical Characterizations

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Abstract

Hierarchically ordered, honeycomb-like nanoporous TiO2 electrodes are prepared by a simple electrochemical anodization process using ammonium fluoride dissolved in ethylene glycol as an electrolytic medium. Formation of hexagonally arranged nanopores along with the tubular structure and anatase crystalline phase of TiO2 is confirmed by field emission scanning electron microscope (FESEM) and X-ray diffraction (XRD) studies. Further, these nanoporous TiO2 electrodes are employed as a substrate for enzyme (horseradish peroxidase, HRP) immobilization in an attempt to enhance the electron transport across the semiconductor electrode–electrolyte interface. Two different strategies, namely, physical entrapment and covalent linking, are used for anchoring the enzyme. Various parameters such as conductivity, stability, enzyme loading, enzymatic activity, sensitivity, linear range, etc., are investigated by using electrochemical techniques. Structural and morphological analyses of enzyme-modified electrodes are carried out using spectroscopic (UV − vis) and microscopic (AFM) methods. In the case of physical entrapment, a simple drop casting method of HRP solution on the nanoporous TiO2 electrodes is used in contrast to chemical linking method where a monolayer of 3-aminopropyltrimethoxy silane (APTMS) is formed initially on TiO2 followed by HRP immobilization using an amide coupling reaction. Interestingly, both of these methods result in anchoring of HRP enzyme, but the amount of enzyme loading and the stability are found to be higher in the covalent linking method. Cyclic voltammetric studies reveal the formation of a well-defined reversible peak for HRP enzyme. Dependence of peak current with the scan rate suggests that HRP enzyme is immobilized and stable and that the overall electron transfer process is predominantly controlled by a diffusion process. Enzymatic activity of HRP is investigated by monitoring the reduction process of hydrogen peroxide by incremental addition using cyclic voltammetry and amperometry techniques, from which several kinetic parameters are determined.

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References

  1. Srinivasan, S., & Chizmadzhev, Y. A. (1985). Comprehensive treatise of electrochemistry, 10. New York: Plenum.

    Book  Google Scholar 

  2. Armstrong, F. A., Heering, H. A., & Hirst, J. (1997). Chemical Society Reviews, 26, 169–179.

    Article  CAS  Google Scholar 

  3. Song, S., Clark, R. A., Bowden, E. F., & Tarlov, M. J. (1993). Journal of Physical Chemistry, 97, 6564–6572.

    Article  CAS  Google Scholar 

  4. Rusling, J. F. (1998). Accounts of Chemical Research, 31, 363–369.

    Article  CAS  Google Scholar 

  5. Boussaad, S., & Tao, N. J. (1999). Journal of the American Chemical Society, 121, 4510–4515.

    Article  CAS  Google Scholar 

  6. Willit, J. L., & Bowden, E. F. (1987). Journal of Electroanalytical Chemistry, 221, 265–274.

    Article  CAS  Google Scholar 

  7. Huang, H., Hu, N., Zeng, Y., & Zhou, G. (2002). Analytical Biochemistry, 308, 141–151.

    Article  CAS  Google Scholar 

  8. Liu, H., & Hu, N. (2003). Analytica Chimica Acta, 481, 91–99.

    Article  CAS  Google Scholar 

  9. Qiao, Z., & Dong, S. (1993). Chinese University Chemistry, 14, 1377–1379.

    CAS  Google Scholar 

  10. Zhao, Q., Gan, Z., & Zhuang, Q. (2002). Electroanalysis, 14, 1609–1613.

    Article  CAS  Google Scholar 

  11. Ahammad, A. J. S., Sarker, S., Rahman, M. A., & Lee, J. J. (2010). Electroanalysis, 22, 694–700.

    Article  CAS  Google Scholar 

  12. Ahammad, A. J. S., Rahman, M. A., Xu, G. R., Kim, S., & Lee, J. J. (2011). Electrochimica Acta, 56, 5266–5271.

    Article  CAS  Google Scholar 

  13. Ahamad, A. J. S., Choi, Y. H., Koh, K., Kim, J. H., Lee, J. J., & Lee, M. (2011). Journal of the Electrochemical Society, 6, 1906–1916.

    Google Scholar 

  14. Xuan, J., Jiang, L. P., & Zhu, J. J. (2010). Chinese Journal of Analytical Chemistry, 38, 513–516.

    Article  CAS  Google Scholar 

  15. Cuendet, P., & Gratzel, M. (1986). Bioelectrochemistry and Bioenergetics, 16, 125–133.

    Article  CAS  Google Scholar 

  16. Wang, B., Zhang, J. J., Pan, Z. Y., Tao, X. Q., & Wang, H. S. (2009). Biosensors and Bioelectronics, 24, 1141–1145.

    Article  CAS  Google Scholar 

  17. Dave, B. C., Dunn, B., Valentine, J. S., & Zink, J. I. (1994). Analytical Chemistry, 66, 1120–1127.

    Article  Google Scholar 

  18. Bowden, E. F., Hawkridge, F. M., & Blount, H. N. (1980). Bioelectrochemistry and Bioenergetics, 7, 447–457.

    Article  Google Scholar 

  19. Ru, J., Du, J., He, H. X., & Lu, X. Q. (2013). Chinese Journal of Analytical Chemistry, 41, 1249–1253.

    Article  CAS  Google Scholar 

  20. Chaplin, M. F., & Bucke, C. (1990). Enzyme technology. Cambridge: Cambridge University Press.

    Google Scholar 

  21. Armstrong, F. A., & Wilson, G. S. (2000). Electrochimica Acta, 45, 2623–2645.

    Article  CAS  Google Scholar 

  22. Liu, S. Q., & Ju, H. X. (2002). Analytical Biochemistry, 307, 110–116.

    Article  CAS  Google Scholar 

  23. Qian, L., & Yang, X. (2006). Talanta, 68, 721–727.

    Article  CAS  Google Scholar 

  24. Wang, L., & Wang, E. (2004). Electrochemistry Communications, 6, 225–229.

    Article  CAS  Google Scholar 

  25. Wang, J., Wang, L., Di, J., & Tu, Y. (2009). Talanta, 77, 1454–1459.

    Article  CAS  Google Scholar 

  26. Hiroyuki, O. (1998). Electrochimica Acta, 43, 1581–1587.

    Article  Google Scholar 

  27. Dunford, H. B. (1991). In J. Eerse, K. E. Everse, & M. B. Grisham (Eds.), Peroxidases in chemistry and biology (p. 1). Boca Raton: CRC.

    Google Scholar 

  28. Creighton, T. E. (1984). Proteins—structure and molecular properties. New York: Freeman.

    Google Scholar 

  29. Lehninger, A. I., Nelson, D. L., & Cox, M. M. (1993). Principles of biochemistry (2nd ed.). New York: Worth.

    Google Scholar 

  30. Ruzgas, T., Csoregi, E., Emnes, J., Gorton, L., & Marko-Varga, G. (1996). Analytica Chimica Acta, 330, 123–138.

    Article  CAS  Google Scholar 

  31. Ekanayake, E. M. I. M., Preethichandra, D. G. M., & Kaneto, K. (2008). Sensors and Actuators B Chemical, 132, 166–171.

    Article  CAS  Google Scholar 

  32. Che, X., Yuan, R., Chai, Y., Ma, L., Li, W., & Li, J. (2009). Microchimica Acta, 167, 159–165.

    Article  CAS  Google Scholar 

  33. Huang, J. L., & Tsai, Y. C. (2009). Sensors and Actuators B Chemical, 140, 267–272.

    Article  CAS  Google Scholar 

  34. Tripathi, V. S., Kandimalla, V. B., & Ju, H. (2006). Biosensors and Bioelectronics, 21, 1529–1535.

    Article  CAS  Google Scholar 

  35. Zhou, K., Zhu, Y., Yang, X., Luo, J., Li, C., & Luan, S. (2010). Electrochimica Acta, 55, 3055–3060.

    Article  CAS  Google Scholar 

  36. Zhou, K., Zhu, Y., Yang, X., & Li, C. (2011). Electroanalysis, 23, 862–869.

    Article  CAS  Google Scholar 

  37. Zhiguo, G., Shuping, Y., Zaijun, L., Xiulan, S., Guangli, W., Yinjun, F., et al. (2011). Analytica Chimica Acta, 701, 75–80.

    Article  Google Scholar 

  38. Lei, C. X., Wang, H., Shen, G. L., & Yu, R. Q. (2004). Electroanalysis, 16, 736–740.

    Article  CAS  Google Scholar 

  39. Zhong, H., Yuan, R., Chai, Y., Li, W., Zhang, Y., & Chengyan, W. (2011). Bioprocess and Biosystems Engineering, 34, 923–930.

    Article  CAS  Google Scholar 

  40. Curulli, A., Cusma, A., Kaciulis, S., Padeletti, G., Pandolfi, L., Valentini, F., et al. (2006). Surface and Interface Analysis, 38, 478–481.

    Article  CAS  Google Scholar 

  41. Si, P., Ding, S., Yuan, J., Lou, X. W., & Kim, D. H. (2011). ACS Nano, 5, 7617–7626.

    Article  CAS  Google Scholar 

  42. Xu, M., Da, P., Wu, H., Zhao, D., & Zheng, G. (2012). Nano Letters, 12, 1503–1508.

    Article  CAS  Google Scholar 

  43. Tang, J., Wang, Y., Li, J., Da, P., Geng, J., & Zheng, G. (2014). Journal of Materials Chemistry A, 2, 6153–6157.

    Article  CAS  Google Scholar 

  44. Paulose, M., Prakasam, H. E., Varghese, O. K., Peng, L., Popat, K. C., Mor, G. K., et al. (2007). Journal of Physical Chemistry C, 111, 14992–14997.

    Article  CAS  Google Scholar 

  45. Yoriya, S., & Grimes, C. A. (2011). Journal of Materials Chemistry, 21, 102–108.

    Article  CAS  Google Scholar 

  46. Hanzu, I., Djenizian, T., & Knauth, P. (2011). Journal of Physical Chemistry C, 115, 5989–5996.

    Article  CAS  Google Scholar 

  47. Baram, N., & Ein-Eli, Y. (2010). Journal of Physical Chemistry C, 114, 9781–9790.

    Article  CAS  Google Scholar 

  48. Murphy, A. B. (2007). Solar Energy Mater. Solar Cells, 91, 1326–1337.

    Article  CAS  Google Scholar 

  49. Anderson, C., & Bard, A. J. (1997). Journal of Physical Chemistry B, 101, 2611–2616.

    Article  CAS  Google Scholar 

  50. Yu, J., & Yu, X. (2008). Environmental Science and Technology, 42, 4902–4907.

    Article  CAS  Google Scholar 

  51. Yi, X., Xian, J. H., & Yuan, C. H. (2000). Analytical Biochemistry, 278, 22–28.

    Article  CAS  Google Scholar 

  52. Ferri, T., Poscia, A., & Santucci, R. (1998). Bioelectrochemistry and Bioenergetics, 45, 221–226.

    Article  CAS  Google Scholar 

  53. Frew, J. E., Harmer, M. A., Hill, H. A. O., & Libor, S. I. (1986). Journal of Electroanalytical Chemistry, 201, 1–10.

    Article  CAS  Google Scholar 

  54. Gu, B. X., Xu, C. X., Zhu, G. P., Liu, S. Q., Chen, L. Y., Wang, M. L., et al. (2009). Journal of Physical Chemistry B, 113, 6553–6557.

    Article  CAS  Google Scholar 

  55. Dimcheva, N., & Horozova, E. (2005). Analytical and Bioanalytical Chemistry, 382, 1374–1379.

    Article  CAS  Google Scholar 

  56. Polsky, R., Harper, J. C., Dirk, S. M., Arango, D. C., Wheeler, D. R., & Brozik, S. M. (2007). Langmuir, 23, 364–366.

    Article  CAS  Google Scholar 

  57. Zhang, Y., Liu, L., Xi, F., Wu, T., & Lin, X. (2010). Electroanalysis, 22, 277–285.

    Article  CAS  Google Scholar 

  58. Kafi, A. K. M., Wu, G., & Chen, A. (2008). Biosensors and Bioelectronics, 24, 566–571.

    Article  CAS  Google Scholar 

  59. Zeng, Y. L., Huang, H. W., Jiang, J. H., Tian, M. N., & Li, C. X. (2007). Analytica Chimica Acta, 604, 170–176.

    Article  CAS  Google Scholar 

  60. Xua, Q., Zhua, J. J., & Hu, X. Y. (2007). Analytica Chimica Acta, 597, 151–156.

    Article  Google Scholar 

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Acknowledgments

The authors acknowledge the funding from the Department of Science and Technology (DST), India, through Fast Track Scheme for Young Scientists with project number GAP 16/10 for carrying out this research work. Central Instrumentation Facility (CIF) of CSIR–CECRI, Karaikudi is also acknowledged for providing necessary characterization facilities.

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  1. Electrodics and Electrocatalysis (EEC) Division, CSIR—Central Electrochemical Research Institute (CSIR–CECRI), Karaikudi, 630006, Tamilnadu, India

    E. T. Deva Kumar & V. Ganesh

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  1. E. T. Deva Kumar
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  2. V. Ganesh
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Correspondence to V. Ganesh.

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Deva Kumar, E.T., Ganesh, V. Immobilization of Horseradish Peroxidase Enzyme on Nanoporous Titanium Dioxide Electrodes and Its Structural and Electrochemical Characterizations. Appl Biochem Biotechnol 174, 1043–1058 (2014). https://doi.org/10.1007/s12010-014-0999-7

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  • DOI: https://doi.org/10.1007/s12010-014-0999-7

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