Another parameter we examined to improve the analytical performances was the concentration of DI. As shown in Figure , the concentration of the reducing enzyme was found to have a considerable impact on the nonspecific autocatalytic response, dramatically accelerating the nonspecific t1/2 value with the rise of DI concentration, an effect which suggests that the enzyme slowly catalyzes the reaction between NADH and O2 to produce O2 •-or H2O2. These results echo the residual NADH oxidase activity (< 0.01%) reported by the enzyme supplier and which would be related to the presence of traces of NADH oxidase in the purified diaphorase samples. However, another possibility that cannot be ruled out is that DI has an intrinsic residual NADH oxidase activity. Indeed, one can assume that the reaction with O2 is not necessarily inexistent even though this flavoenzyme is known to not accept O2 as an electron acceptor. By gradually decreasing the concentration of DI, we can see in Figure that the value of t1/2 increases until it reaches a maximum before slightly decreasing at the lowest concentration (graph B in Figure ). This indicates that by lowering the concentration of DI, the nonspecific contribution associated to the residual NADH oxidase activity is reduced. This can therefore be exploited to improve the specific vs. nonspecific response, especially if the enzyme reduction rate of the naphthoquinone in Loop 2 continues to be not rate limiting. This latter attribute can be easily verified from the S-shape of the autocatalytic response. Indeed, if the kinetic profiles are similar regardless of the enzyme concentration, it suggests that the naphthoquinone reduction is not rate limiting. On the other hand, when the rate of the naphthoquinone reduction becomes too slow, as illustrated for the lowest enzyme concentration (i.e. 0.1 nM in Figure ), the kinetic trace presents a more spreadout autocatalytic response, signing thus the slowing down of Loop 2. The kinetic gain also observed at the lowest enzyme concentrations can be explained from the redox cycling (Loop 2) in Scheme 3, which tends to be inhibited at high DI concentration by forcing the naphthoquinone to be rapidly doubly reduced, thus bypassing the formation of semi-naphthoquinone indispensable to sustain the free radical chain reaction. This inhibitory effect is relaxed when the enzyme concentration becomes sufficiently low, which finally leads to a slightly faster autocatalytic reaction. Considering the importance of the enzyme concentration on the nonspecific response, we reproduced the autocatalysis experiments at pH 8.5 with a DI concentration 100 times lower, i.e. 0.1 nM. The results in Figure show a significant gain in the H2O2 detection limit, allowing now to detect as low as 15 nM H2O2. Compared to the previous experiments performed at a higher enzyme concentration, this corresponds to a 10-fold gain in LOD (compare Figures and).