Abstract
It is rather difficult to achieve the direct electrochemistry of nitrate reductase (NR) as it is a complex homodimeric enzyme. However, in this study, the direct electron transfer between NR's redox centers and the electrode surface was achieved with the aid of aminated graphene sheets (am-GSs) which could immobilize NR stably and control the orientation of the enzyme molecules on the surface of the modified electrode through electrostatic attractions. Moreover, when the gold nanoparticles (AuNPs) which could act as electronic wire were introduced to the modified electrode, the NR-based enzymatic reduction of nitrate was promoted and a sensitive electrochemical response regarding the electrochemical reduction of nitrate could be obtained at the NR/AuNPs/am-GSs/GC electrode. Under optimized conditions, a wide linear range from 1.0 × 10(-6) mol L(-1) to 2.0 × 10(-3) mol L(-1) was acquired with a low detection limit of 7 × 10(-7) mol L(-1) (S/N = 3). The biosensor was successfully employed to determine the total nitrogen in environmental water samples and the results were in good accordance with those obtained by ultraviolet-visible spectrophotometry.