Extreme pH values resulted in the undesired formation of Prussian blue (Fig. ). At pH = 5, we fitted the current responses using i = imax (1-exp(-kobs•t)), where the fitting parameters of imax and kobs correspond to the maximum current and observed reaction rate constant, respectively, and found that the fitted kobs values are nearly independent of creatinine concentration, indicating pseudo-first order kinetics (Fig. ). The observed reaction rates increased with temperature following the Arrhenius equation (Figs. and), with an activation energy, Ea, of 10.9±0.4 kJ mol -1 and a pre-exponential parameter, A, of 26.0±1.4 s -1 . Using these values, we created a reaction yield map as a function of time and temperature (Fig. ). For example, at 65°C, one can reach a reaction yield of >95% after 25 minutes. We then carried out mechanistic analysis of the reaction, starting with an assessment of the role of ferricyanide in the rate-limiting step (Figs. and). The observed reaction rates were linearly proportional to the initial ferricyanide concentration (the first-order contribution), indicating that the rate-limiting step involves a bimolecular reaction between creatinine and an excess of ferricyanide (Fig. ). We also investigated the impact of creatinine concentration (Fig. ). The response reveals a linear dependence up to 27.5 mM, beyond which the current suddenly drops due to CE limitations. By correlating the sampled current values in the linear region with the ferrocyanide oxidation signals calibrated in Fig. , we estimate that on average 3.80±0.11 ferricyanide molecules reacted with one creatinine molecule (Fig. ). In other words, the total reaction corresponds to the oxidation of two chemical bonds per creatinine molecule, leading to the reduction of four ferricyanide molecules. In light of these findings, we hypothesize that the kinetics involve four sequential steps in which one creatinine molecule is progressively oxidized by one ferricyanide molecule at each step. The analysis presented here not only identifies the optimal reaction conditions, but also offers a rationale for the system limitation. More specifically, one must keep the ferricyanide concentration at least eight times (2×4) higher than the maximum creatinine concentration to be detected, taking into account the dilution factors when implemented in a sample matrix (such as urine).