To initiate the assay, 50 μL of standard solutions and urine samples was pipetted into microwells. Each sample underwent triplicate assays and was tested in parallel with creatinine standard solutions. The sample zone of each enz-PAD was dipped into wells of the standard or urine solutions to allow creatinine in the sample to react with the pre-impregnated R1 and R2 reagents via the natural capillary force for 4 min. Meanwhile, the water front usually reached and covered the detection zone. Then, the enzPAD devices were removed from the sample wells, and the colour was allowed to completely develop using air-drying in the detection zone for another 7 min (Fig. 2). Finally, the enz-PAD was imaged using a scanner (HP Deskjet F370 All-in-One scanner) at 600 dpi resolution in Jpeg format.The detection zone, a fixed size corresponding to 70 90 pixels in rectangular area on the enzPAD, was selected for measuring the colour intensity. An average intensity value of the rectangular area was obtained from the histogram panel of RGB channel in the Adobe Photoshop CS2 programme.