Following the manufacturer instructions, a 2:1 ratio of R1–R2 is recommended for a reaction mixture of the enzymatic creatinine assay. Therefore, the same proportion of both reagents was utilised in this study. The PADs were placed on a cleaned lab bench, in which the printed pattern was side up. Then, the reagents were applied to the desired areas using a pipetting technique, in which 5 mL of R1 and 2.5 mL of R2 were adsorbed on the desired regions separately (Fig. 1b). To carry the enz-PAD, it was touched only at the hydrophobic holder zone. Next, the enz-PADs were allowed to air dry at room temperature, and a vacuum sealer (SINBO™) was used to seal the enz-PADs in a plastic bag for storage at 4 °C until future use. Based on the 1–3 mL dead volume of the obtained reagents, 200–600 enz-PADs were fabricated. This, demonstrated a very low or near zero-cost for this paper device.