Simultaneous analysis of multiple adulterants in milk using microfluidic paper-based analytical devices

This study reports the simultaneous colorimetric detection of urea, H₂O₂, and pH in milk samples using microfluidic paper-based analytical devices (μPADs) fabricated through a craft cutter printer. Paper-based devices were designed to contain three detection zones interconnected to a sampling zone by microfluidic channels. Colorimetric analysis was performed using images digitalized through an office scanner. The volumes of chromogenic and sample solutions were optimized, and the best colorimetric performance was achieved by adding 0.5 and 10 μL into detection and sampling zones, respectively. Simultaneous assays were then carried out, and the recorded responses revealed a linear behavior in the concentration ranges from 0-30.0 mmol L^-1, 0-10.0 mmol L^-1 and 6.0-9.0 for urea, H₂O₂ and pH, respectively. The limit of detection values obtained for urea and H₂O₂ were 2.4 mmol L^-1 and 0.1 mmol L^-1, respectively. For pH measurements, colorimetric assay allowed the monitoring of solution pH with a resolution of 0.25 units. The use of μPADs to detect target adulterants exhibited suitable reproducibility (RSD ≤ 6.0%), accuracy (91-102%) and no cross-reaction occurrence. When compared to reference techniques, colorimetric assays did not reveal a significant difference at a confidence level of 95%. As a proof-of-concept, the feasibility of the proposed approach was successfully demonstrated through the analysis of potential adulterants in sixteen milk samples, which were tested without any pretreatment requirement. Based on the achievements, μPADs in conjunction with colorimetric measurements emerge as a powerful tool for rapid screening of potential adulterants in milk.