Urinalysis is widely used in routine medical checkups. Conventionally, colorimetric paper strips currently available on the market are applied. Although they allow to rapidly obtain results, their use is often not straightforward, because a timed readout is required, followed by comparison with a color chart. D-μPADs (distance-based microfluidic paper-based analytical devices) have gained attention for enabling highly intuitive signal readout. During liquid flow through a paperfluidic channel, target analytes are sequentially depleted from the sample through reaction with colorimetric indicators and/or adsorption to the paper surface, so that the length of color-changed sections correspond to the analyte content. Therefore, D-μPADs are expected to solve the problems of existing urinalysis tests. The final goal is the development of D-μPADs for the simultaneous detection of urinary protein (albumin), glucose and hemoglobin. Colorimetric distance-based calibration curves were acquired for all analytes in artificial urine matrix containing electrolytes and organic compounds. For quantitative assays, evaluation of color-changed distances was conducted by computer software. The first cases of distance-based albumin, glucose and hemoglobin readouts in relatively complex artificial urine matrix are reported. For albumin detection, the color uniformity of the colorimetric indicator was improved by paper surface modification with poly(4-styrenesulfonic acid), an anionic polymer. For glucose detection, signal visibility was significantly improved by paper surface modification with chitosan, a biopolymer that works as immobilization support for enzymes. Finally, we evaluated the effect of urinary components on the assay results. It was confirmed that D-μPADs for the hemoglobin assay require a treatment to reduce the effect of albumin adsorption on the paper substrate.
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