In this study, determination of creatinine is based on an enzymatic method. Therefore, the pH of an assay reaction influences enzymatic activity and the colour intensity development. In our enz-PAD platform, urine samples represent a relatively large volume compared with the reagent that was impregnated on the paper. Therefore, each urine sample should be adjusted to the same optimised pH before the assay is performed. The average pH of healthy urine samples ranges from 6.5 to 8.0. In the experiment, a variety of buffers with pH ranging from 6.5 to 9.0 was used to dilute the samples and to determine the optimal pH. The results demonstrated in Fig. 3 show that the colour intensity increased when pH of the sample increased. However, a pH of samples higher than 7.5 did not influence the enzyme activity, such as for the use of potassium phosphate buffer, and therefore, a colour intensity plateau was observed. Clearly, if the pH of the sample was higher than 7.8, the colour intensity was dramatically decreased. Therefore, in this study, a 100 mM potassium phosphate buffer with a pH of 7.5 was selected to dilute samples before the enz-PAD analysis.