Engineered detection zone to enhance color uniformity on paper microfluidics fabricated via Parafilm®-heating-laser-cutting

In this article, we first report a simple fabrication technique to create Parafilm®-laminated microfluidic paper-based analytical devices (µPADs) with a high resolution and then introduce novel engineered detection zones (D-zones) to enhance color uniformity without any chemical modifications. The Parafilm®-heating-laser cutting fabrication method comprises of three steps: 1) flattening a Parafilm® layer on paper, 2) heating in an oven at 90 °C for 30 min, and 3) CO2 laser ablation of the patterns. This method enabled laminated µPADs with a minimum barrier width of 172 ± 15 µm, with resistance to various organic solvents and surfactant solutions. Using this technique, µPADs with two types of engineered D-zones were fabricated. The multi-inlet D-zone had four micro-inlets (<500 µm width), enabling sample entrance into the D-zone from four directions simultaneously towards the center. The segmented D-zone had four segments each with its own micro-inlet allowing for separate reactions to occur in miniaturized segments simultaneously. Using an enzymatic glucose assay for the colorimetric reaction, the multi-inlet and segmented D-zones significantly decreased color gradient from 28.77 ± 1.49 to 12.35 ± 1.18 and 8.95 ± 0.98 %, respectively, compared to the conventional devices. We further used the µPADs with segmented D-zones for the detection of glucose in artificial urine in the clinically relevant range of 2–50 mM, where R2 > 0.99 of the fitted curve showed an excellent correlation between the generated signals and the glucose concentrations. Also, compared to the conventional devices, the engineered D-zones showed improved precision and reproducibility and a better linear range. Finally, spiked real human urine samples were tested, demonstrating the applicability of the engineered devices for real-world applications. Overall, with a simple fabrication method and a novel design for color uniformity, this paper contributes to the advancement of µPADs and paves the way towards commercialization.