A Laminated Microfluidic Paper-Based Analytical Device Based on Image Analysis Using Optimized Color Space for Simultaneous Multiplexed Quantification of Chemical Water Quality Parameters

Abstract Water contamination remains a critical global challenge, underscoring the need for portable, low-cost, and user-friendly tools for on-site monitoring, particularly in resource-limited settings. This work presents a laminated multilayered wax-printed microfluidic paper-based analytical device (μPAD) engineered to provide rapid, multiplexed quantification of three key water quality indicators, covering both chemical contaminations represented by (NO2− and Fe2+) and physical water quality assessed through total hardness (Ca2+ and Mg2+) in a single test. The lamination-based assembly ensures strong interlayer adhesion, uniform contact, and stable capillary-driven flow. This design effectively prevents reagent leakage, backflow, and layer separation, which are common limitations in conventional paper-based devices. This study offers the first systematic evaluation of ten color spaces for image-based quantification of a μPAD, enabling evidence-based selection of optimal colorimetric parameters for enhanced analytical performance. The platform supports rapid operation with a 5 min detection time and achieves limits of detection of 0.07 ppm (NO2−), 0.34 ppm (Fe2+), 6.0 ppm (Mg2+), and 9.0 ppm (Ca2+), outperforming other recently reported platform results and aligning with regulatory requirements. Robust selectivity was demonstrated across diverse real-water matrices; recovery values ranged from 77.3% to 126%, consistent with accepted criteria for trace-level analysis. Collectively, these results highlight the device capability to deliver accurate, reliable, and comprehensive water quality assessment in a single test, offering a scalable solution for field deployment.