Microchimica Acta

, Volume 182, Issue 11–12, pp 1949–1956 | Cite as

Enhanced amperometric response of a glucose oxidase and horseradish peroxidase based bienzyme glucose biosensor modified with a film of polymerized toluidine blue containing reduced graphene oxide

  • Fang WangEmail author
  • Wencheng Gong
  • Lili Wang
  • Zilin ChenEmail author
Original Paper


Reduced graphene oxide (RGO) was used to construct a bienzyme biosensor containing horseradish peroxidase (HRP) and glucose oxidase (GOx). A poly(toluidine blue) (pTB) film containing RGO acted as both enzyme immobilization matrix and electron transfer mediator. The bienzyme biosensor was characterized by electrochemical techniques and displays a highly sensitive amperometric response to glucose and hydrogen peroxide (H2O2) at a potential as low as −0.1 V (vs. SCE). It is shown that use of RGO causes a strong enhancement on the amperometric responses. H2O2 formed by the action of GOx in the presence of oxygen can be further reduced by HRP in the pTB film contacting the RGO modified electrode. In the absence of oxygen, glucose oxidation proceeds by another mechanism in which electron transfer occurs from GOx to the electrode and with pTB acting as the mediator. Amperometric responses to glucose and H2O2 follow Michaelis-Menten kinetics. The experimental conditions were optimized, and under these conditions glucose can be determined in the 80 μM to 3.0 mM range with a detection limit of 50 μM. H2O2, in turn, can be quantified in up to 30.0 μM concentration with a detection limit of 0.2 μM. The bienzyme biosensor is reproducible, repeatable and stable. Finally, it has been successfully applied to the determination of glucose in plasma samples.

Graphical Abstract

Schematic representation of glocuse detection at GCE/RGO/pTB-HRP-GOx.


Reduced graphene oxide Bienzyme biosensor Horseradish peroxidase Glucose oxidase Toluidine blue Amperometry; 



This work was supported by the National Scientific Foundation of China (NSFC NOs. 21375101 and 60801020), the Fundamental Research Funds for the Central Universities (2042014kf0295), and Natural Science Foundation of Hubei Province (ZRY2014000492), Wuhan Science and Technology Bureau (No: 20140601010057).


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Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  1. 1.Wuhan University School of Pharmaceutical ScienceWuhanChina
  2. 2.State Key Laboratory of Transducer TechnologyChinese Academy of SciencesBeijingChina

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