| The main purpose of this work is to study the fabrication of the third-generation electrochemical biosensors by using proper method to explore the novel materials in the fabrication of bioelectrochemical interface and evaluating the resulting biosensors. The open and regular structure of layered nanomaterials provides new thoughts for the immobilization of redox proteins. In this work, a sensitive biosensor was fabricated by incorporating myoglobin（Mb） within layered Mo S2-graphene and Nafion composite film. The main contents of this work are given as follows:The mixture of Mo S2 and graphene dispersion was cast on the electrode surface. Then, Mb and Nafion were decorated on the surface of layered Mo S2-graphene composite modified electrode sequentially, and formed a stable film. The layered films were characterized by scanning electron microscopy（SEM）, Fourier transform infrared spectroscopy（FT-IR）, UV-vis spectrum（UV-vis） and various electrochemical techniques. UV-vis spectrum demonstrated that Mb retained its secondary structure similar to its native states after incorporated within layered Mo S2-graphene/Nafion film. Effective direct electron transfer（DET） of Mb was realized with the presence of a pair of well defined and nearly reversible peaks at about-0.38 V vs saturated calomel electrode（SCE） in p H 7.0 buffers. The electron transfer rate constant（ks） obtained by fitting the data of square wave votammogram（SWV） with nonlinear regression analysis was about 60.0 s-1, this may be due to the synergistic effect of Mo S2 and graphene. The formal potential of the Mb FeIII/FeII couple in layered Mo S2-graphene/Nafion films shifted linearly between p H 4.0 and 11.0 with a slope of-50.0 m V p H-1, suggesting that one proton transfer is coupled to each electron transfer in the electrochemical reaction. Moreover, the biosensor exhibited excellent analytical performance for hydrogen peroxide（H2O2）, oxygen（O2）, sodium nitrite（Na NO2） and trichloroacetic acid（TCA）. These results demonstrated that the electrochemical biosensor fabricated by this method has high sensitivity, good selectivity and stable catalytic activities. The obtained results demonstrated that layered Mo S2-graphene composites were promising for the fabrication of novel biosensors and electronic devices.
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