Nanoliter-Fabricated Paper-Based Colorimetric Lateral Flow Strip for Urea Detection

A nanoliter-scale fabrication method was applied to construct a colorimetric lateral flow strip for urea detection (Urea-CLFS). The device involves two main papers: a nitrocellulose membrane (NC-Mb) for urease enzyme immobilization and chromatography paper (CH-PP) containing a phenol red indicator. Urea-CLFS is a tool for detecting urea that is based on enzyme catalysis and the change in color of phenol red when urea is present. The Urea-CLFS fabrication was made possible by the minimal amount of nanoliters used in reagent consumption. The use of small arrays of phenol red dots provides a higher response result compared to single dots applied on CH-PP. To find the most effective design, it analyzed how urease was aligned on NC-Mb horizontally and vertically. According to our findings, the vertical alignment of the urease enzyme on NC-Mb leads to a prolonged reaction time, which leads to higher product production. The optimization process included optimizing various parameters, including the layer number of phenol red on CH-PP, phenol red concentration, urease concentration, reaction time, and sample volume. Under optimal conditions, the Urea-CLFS provided a linear range of 0.25–8.0 mmol L−1 with an LOD of 0.34 mmol L−1, which is sufficient for human health diagnostics. The accuracy of the Urea-CLFS was demonstrated by the recovery of the human urine sample between 95 ± 3% and 103 ± 3% (n = 3).