It is very important to select the optimum nicking reaction time because different reaction times of the Nt.AlWI might cause differ-ent responses. The i of MB tags was proportional to the extent of the nicking reaction which was dependent on the incubationtime in Nt.AlWI solution. When the DNA biosensor was incubated in 25 L of 0.2 U L−1Nt.AlWI solution and the experiment was done at 50◦C, the i value increased significantly with increasing nicking reaction time and approached a value that represents saturation of the enzyme activity after 120 min. Beyond the nicking reaction time, the i no longer increased obviously, as shown inFig. 5. Taking this into account, 120 min was chosen for the nicking reaction time.