Unless otherwise stated, Whatman No. 3 filter paper was used to fabricate the enz-PADs because the paper has appropriate pore sizes and thickness suitable for our approach. The Whatman No. 3 filter paper was designed to consist of five zones, which included two reagent adsorption zones (R1; 4 10 mm2 and R2; 4 7 mm2), a sample dipping zone (4 5 mm2), a detection zone (4 3 mm2) and a hydrophobic holder zone (4 15 mm2). It is necessary to adsorb the R1 and R2 enzyme reagents on separated zones for the following reasons. The existing endogenous creatine in the sample needs to be eliminated in the first step during the contact with R1 before a reaction with creatininase is started in R2. Additionally, the generated H2O2 from the creatinase and sarcosine oxidase reaction needs to be destroyed using the catalase in the R1 zone. The, the catalase is inhibited with sodium azide when it flows to R2, so the actual H2O2 that is generated from the creatinine reaction does not interfere. The patterned papers were printed using an HP LaserJet P1102 printer and then, were cut into small rectangular shapes (4 40 mm2). Furthermore, a hydrophobic holder zone was created by dipping one end of the paper into a melted wax chamber at 10873 °C for 3 s. In addition, the wax holder zone acted as abarrier for the accumulating quinone imine products. The enz-PAD design is shown in Fig. 1.