A 3D Microfluidic Paper-Based Analytical Device with Smartphone-Based Colorimetric Readout for Phosphate Sensing

In this work, a 3D microfluidic paper-based analytical device (3D-µPAD) was developed for the smartphone-based colorimetric determination of phosphate in environmental samples. The assay relied on the formation of a blue-colored product (molybdenum blue) in the detection area of the 3D-µPAD upon reduction of the heteropolyacid H₃PMo₁₂O₄₀ formed in the presence of phosphate. A number of experimental parameters were optimized, including geometric aspects of 3D-µPADs, digitization and image processing conditions, the amount of chemicals deposited in specific areas of the 3D-µPAD, and the reaction time. In addition, the stability of the device was evaluated at three different storage temperatures. Under optimal conditions, the working range was found to be from 4 to 25 mg P/L (12-77 mg PO₄^-3/L). The limits of detection (LOD) and quantification (LOQ) were 0.015 mg P/L and 0.05 mg P/L, respectively. The repeatability and intermediate precision of a 5 mg P/L standard were 4.8% and 7.1%, respectively. The proposed colorimetric assay has been successfully applied to phosphorous determination in various waters, soils, and sediments, obtaining recoveries in the range of 94 to 107%. The ready-to-use 3D-µPAD showed a greener profile than the standard method for phosphate determination, being affordable, easy-to-use, and suitable for citizen science applications.