When the DNA biosensor was incubated in 25 L of 0.2 U L−1 Nt.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 in Fig. 5. Taking this into account, 120 min was chosen for the nicking reaction time.