A Novel Label-Free Immunosensor Based on Electrochemically Reduced Graphene Oxide for Determination of Hemoglobin A1c

M. Özge Karaşallı M. Özge Karaşallı ,  Derya Koyuncu Zeybek Derya Koyuncu Zeybek
Российский электрохимический журнал
Abstract / Full Text

In this work, a novel immunosensor has been developed using an electrochemically reduced graphene oxide (ERGO) modified glassy carbon electrode (GCE) for sensitive and label-free detection of glycated hemoglobin (HbA1c) used as the gold standard marker for glycemic control. The prepared immunosensor, using ferrocyanide/ferricyanide as the mediator, ensures an uncomplicated and economic method for the detection of HbA1c. The proposed immunosensor can directly record changes in the electrochemical signals when the immunoreaction occurs on the sensor surface. The specific antibody-antigen immunoreaction resulted in a reduction in the amperometric response of the mediator that diffused at the electrode surface. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were utilized to investigate the electrochemical characteristics of the immunosensor. Calibration curve was plotted via differential pulse voltammetry (DPV) technique and a linear relationship was obtained between the DPV response and HbA1c concentrations from 1 to 25%. Under the optimized condition, the GCE/ERGO/HbA1cAb immunosensor supplied high selectivity and sensitivity for HbA1c detection in real serum samples.

Author information
  • Kütahya Dumlupınar University, Faculty of Science and Arts, Department of Biochemistry, Kütahya, Turkey

    M. Özge Karaşallı &  Derya Koyuncu Zeybek

  1. Pundir, C.S. and Chawla, S., Determination of glycated hemoglobin with special emphasis on biosensing methods, Anal. Biochem., 2014, vol. 444, p. 47.
  2. Yazdanpanah, S., Abdolrahim, M., Rabie, M., and Tayebi, L., Glycated hemoglobin-detection methods based on electrochemical biosensors, Trends Anal. Chem., 2015, vol. 72, p. 53.
  3. Tanaka, T., Izawa, K., Okochi, M., Lim, T.-K., Shugo, Watanabe, Harada, M., and Matsunaga, T., On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level, Anal. Chim. Acta, 2009, vol. 638, p. 186.
  4. Tanaka, T., Tsukube, S., Izawa, K., Okochi, M., Lim, T.-K., ShugoWatanabe, Harada, M., and Matsunaga, T., Electrochemical detection of HbA1c, a maker for diabetes, using a flow immunoassay system, Biosens. Bioelectron., 2007, vol. 22, p. 2051.
  5. Siren, H., Laitinen, P., Turpeinen, U., and Karppinen, P., Direct monitoring of glycohemoglobin A in the blood samples of 1c diabetic patients by capillary electrophoresis comparison with an immunoassay method, J. Chromatogr., 2002, vol. 979, p. 201.
  6. Little, R.R. and Roberts, W.L., A review of variant hemoglobins interfering with hemoglobin A1c measurement, J. Diabetes Sci. Technol., 2009, vol. 3, no. 3, p. 446.
  7. Danilowicz, C. and Manrique, J.M., A new self-assembled modified electrode for competitive immunoassay, Electrochem. Commun., 1999, vol. 1, p. 22.
  8. Li, R., Wu, D., Li, H., Xu, C., Wang, H., Zhao, Y., Cai, Y., Wei, Q., and Du, B., Label-free amperometric immunosensor for the detection of human serum chorionic gonadotropin based on nanoporous gold and graphene, Anal. Biochem., 2011, vol. 414, p. 196.
  9. Yang, L., Zhao, H., Fan, S., Deng, S., Lv, Q., Lin, J., and Li, C.-P., Label-free electrochemical immunosensor based on gold-silicon carbide nanocomposites for sensitive detection of human chorionic gonadotrophin, Biosens. Bioelectron., 2014, vol. 57, p. 199.
  10. Alonso-Fernándeza, M., Mancera-Romero, J., Mediavilla-Bravo, J.J., Comas-Samper, J.M., López-Simarro, F., Pérez-Unanua, M.P., and Iturralde-Iriso, J., Glycemic control and use of A1c in primary care patients with type 2 diabetes mellitus, Prim. Care Diabetes, 2015, vol. 9, p. 385.
  11. Meyer, J.C., Geim, A.K., Katsnelson, M.I., Novoselov, K.S., Booth, T.J., and Roth, S., The structure of suspended graphene sheets, Nature, 2007, vol. 46, p. 60.
  12. Yang, J., Deng, S., Lei, J., Ju, H., and Gunasekaran, S., Electrochemical synthesis of reduced graphene sheet-AuPd alloy nanoparticle composites for enzymatic biosensing, Biosens. Bioelectron., 2011, vol. 29, no. 1, p. 159.
  13. Hua, L., Wu, X., and Wang, R., Glucose sensor based on an electrochemical reduced graphene oxide-poly(l-lysine) composite film modified GC electrode, Analyst, 2012, vol. 137, no. 24, p. 5716.
  14. Bhardwaj, S.K., Mahapatro, A.K., and Basu, T., Benzymatic triglyceride biosensor based on electrochemically reduced graphene oxide, Int. J. Chemtech. Res., 2015, vol. 7, no. 2, p. 858.
  15. Zhang, Y., Zhang, J., Wu, H., Guo, S., and Zhang, J., Glass carbon electrode modified with horseradish peroxidase immobilized on partially reduced graphene oxide for detecting phenolic compounds, J. Electroanal. Chem., 2012, vol. 681, p. 49.
  16. Tabrizi, M.A., Azar, S.J., and Varkani, J.N., Eco-synthesis of graphene and its use in dihydronicotinamide adenine dinucleotide sensing, Anal. Biochem., 2014, vol. 460, p. 29.
  17. Özcan, B. and Sezgintürk, M.K., Graphene oxide based electrochemical label free immunosensor for rapid and highly sensitive determination of tumor marker HSP70, Talanta, 2016, vol. 160, p. 367.
  18. Guerrero, S., Martinez-Garcia, G., Serafin, V., Agui, L., Yanez-Sedeno, P., and Pingarron, J.M., Electrochemical immunosensor for sensitive determination of the anorexigen peptide YY at grafted reduced graphene oxide electrode platforms, Analyst, 2015, vol. 140, no. 22, p. 7527.
  19. Zhou, Y., Dong, H., Liu, L., Hao, Y., Chang, Z., and Xu, M., Fabrication of electrochemical interface based on boronic acid-modified pyrroloquinoline quinine/reduced graphene oxide composites for voltammetric determination of glycated hemoglobin, Biosens. Bioelectron., 2015, vol. 64, p. 442.
  20. Hummers, W.S. and Offeman, R.E., Preparation of graphitic oxide, J. Am. Chem. Soc., 1958, vol. 80, p. 1339.
  21. Pham, T.A., Kim, J.S., Kim, J.S., and Jeong, Y.T., One-step reduction of graphene oxide with L-glutathione, Colloids Surf. Physicochem. Eng. Aspects, 2011, vol. 384, p. 543.
  22. Qiao, L., Wang, X., and Sun, X., A novel label-free amperometric immunosensor based on graphene sheets-methylene blue nanocomposite/gold nanoparticles, Int. J. Electrochem. Sci., 2014, vol. 9, no. 3, p. 1399.
  23. Stankovich, S., Dikin, D.A., Piner, R.D., Kohlhaas, K.A., Kleinhammes, A., Jia, Y., Wu, Y., Nguyen, S.T., and Ruoff, R.S., Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon, 2007, vol. 45, p. 1558.
  24. Wang, J., Study of electrode reactions and interfacial properties, in Analytical Electrochemistry, 3rd ed., John Wiley & Sons, 2006, p. 29. https://doi.org/10.1002/0471790303.ch2
  25. Mishra, S.K., Srivastava, A.K., Kumar, D., Biradar, A.M., and Rajesh, Microstructural and electrochemical impedance characterization of bio-functionalized ultrafine ZnS nanocrystals-reduced graphene oxide hybrid for immunosensor applications, Nanoscale, 2013, vol. 5, no. 21, p. 10494.
  26. Hu, X., Dou, W., and Zhao, G., Electrochemical immunosensor for Enterobacter sakazakii detection based on electrochemically reduced graphene oxide-gold nanoparticle/ionic liquid modified electrode, J. Electroanal. Chem., 2015, vol. 756, p. 43.
  27. Ramanavicius, A., Finkelsteinas, A., Cesiulis, H., and Ramanaviciene, A., Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor, Bioelectrochemistry, 2010, vol. 79, no. 1, p. 11.
  28. Wu, C.-C., Lin, C.-H., and Wang, W.-S., Development of an enrofloxacin immunosensor based on label-free electrochemical impedance spectroscopy, Talanta, 2009, vol. 79, no. 1, p. 62.