To determine the optimal reaction time for enzymatic reaction before capturing the image intensity, 2.5 and 20 mg dL1 creatinine sample solution were tested with the proposed enz-PADs. When the sample solution reached and covered the detection zone, the devices were removed from the sample wells, and the reaction time was immediately recorded. The colour intensity in the detection zone was captured using a scanner for another 15 min at 1-min intervals. As shown in Fig. 4, the colour intensity was developed proportional to time within the first 3–7 min. As the reaction extended longer than 7 min, the colour intensity was slowly developed. As a compromise between the sensitivity of the assay and the assay time, a reaction time of 7 min was selected for the creatinine measurements using enz-PADs in the subsequent experiments