▎ 摘　　要

A novel strategy for fabrication of a bienzyme complex for biosensing based on the construction of a 2-D graphene framework was presented. First, a graphene-chitosan (GR-CS) nanocomposite sheet with excellent electrical conductivity and biocompatibility was prepared and used for enzyme assembly. Then, glucose oxidase (GOD) and horseradish peroxidase (HRP) were selected as model enzymes and immobilized covalently on the different GR-CS nanosheets, respectively. The two enzyme-immobilized sheets were linked together to form a 2-D graphene framework through biospecific interaction of avidin and biotin. Detailed studies indicated that, the GOD/HRP bienzyme complex confined in the 2-D graphene framework have excellent electrochemical activity and high synergistic catalytic activity toward glucose biosensing. More importantly, the synergic catalytic efficiency of the GOD/HRP bienzyme complex for glucose could be regulated by changing the interlayer space of the 2-D graphene framework. Furthermore, the GOD/HRP bienzyme complex for glucose biosensing exhibited good reproducibility and stability, wide linear range of 0.005-5.0 mM, and high sensitivity with LOD of 1.50 mu M, which could be applied to the determination of glucose in human serum samples with reliable results. Therefore, the developed bienzyme complex in the work could offer a platform of constructing biosensors and bioreactor for different substrates. (C) 2015 The Electrpchemical Society. All rights reserved.